@article{irvin_wang_2024, title={Single-molecule fluorescence imaging of DNA maintenance protein binding dynamics and activities on extended DNA}, volume={87}, ISSN={["1879-033X"]}, DOI={10.1016/j.sbi.2024.102863}, journal={CURRENT OPINION IN STRUCTURAL BIOLOGY}, author={Irvin, Elizabeth Marie and Wang, Hong}, year={2024}, month={Aug} }
 @article{liu_pan_kaur_wang_jin_detwiler_opresko_tao_wang_riehn_2023, title={Assembly path dependence of telomeric DNA compaction by TRF1, TIN2, and SA1}, volume={122}, ISSN={["1542-0086"]}, url={https://doi.org/10.1016/j.bpj.2023.04.014}, DOI={10.1016/j.bpj.2023.04.014}, abstractNote={Telomeres, complexes of DNA and proteins, protect ends of linear chromosomes. In humans, the two shelterin proteins TRF1 and TIN2, along with cohesin subunit SA1, were proposed to mediate telomere cohesion. Although the ability of the TRF1-TIN2 and TRF1-SA1 systems to compact telomeric DNA by DNA-DNA bridging has been reported, the function of the full ternary TRF1-TIN2-SA1 system has not been explored in detail. Here, we quantify the compaction of nanochannel-stretched DNA by the ternary system, as well as its constituents, and obtain estimates of the relative impact of its constituents and their interactions. We find that TRF1, TIN2, and SA1 work synergistically to cause a compaction of the DNA substrate, and that maximal compaction occurs if all three proteins are present. By altering the sequence with which DNA substrates are exposed to proteins, we establish that compaction by TRF1 and TIN2 can proceed through binding of TRF1 to DNA, followed by compaction as TIN2 recognizes the previously bound TRF1. We further establish that SA1 alone can also lead to a compaction, and that compaction in a combined system of all three proteins can be understood as an additive effect of TRF1-TIN2 and SA1-based compaction. Atomic force microscopy of intermolecular aggregation confirms that a combination of TRF1, TIN2, and SA1 together drive strong intermolecular aggregation as it would be required during chromosome cohesion.}, number={10}, journal={BIOPHYSICAL JOURNAL}, author={Liu, Ming and Pan, Hai and Kaur, Parminder and Wang, Lucia J. and Jin, Miao and Detwiler, Ariana C. and Opresko, Patricia L. and Tao, Yizhi Jane and Wang, Hong and Riehn, Robert}, year={2023}, month={May}, pages={1822–1832} }
 @article{kaur_lu_xu_irvin_pappas_zhang_finkelstein_shi_tao_yu_et al._2023, title={High-speed AFM imaging reveals DNA capture and loop extrusion dynamics by cohesin-NIPBL}, volume={299}, ISSN={["1083-351X"]}, DOI={10.1016/j.jbc.2023.105296}, abstractNote={3D chromatin organization plays a critical role in regulating gene expression, DNA replication, recombination, and repair. While initially discovered for its role in sister chromatid cohesion, emerging evidence suggests that the cohesin complex (SMC1, SMC3, RAD21, and SA1/SA2), facilitated by NIPBL, mediates topologically associating domains and chromatin loops through DNA loop extrusion. However, information on how conformational changes of cohesin-NIPBL drive its loading onto DNA, initiation, and growth of DNA loops is still lacking. In this study, high-speed atomic force microscopy imaging reveals that cohesin-NIPBL captures DNA through arm extension, assisted by feet (shorter protrusions), and followed by transfer of DNA to its lower compartment (SMC heads, RAD21, SA1, and NIPBL). While binding at the lower compartment, arm extension leads to the capture of a second DNA segment and the initiation of a DNA loop that is independent of ATP hydrolysis. The feet are likely contributed by the C-terminal domains of SA1 and NIPBL and can transiently bind to DNA to facilitate the loading of the cohesin complex onto DNA. Furthermore, high-speed atomic force microscopy imaging reveals distinct forward and reverse DNA loop extrusion steps by cohesin-NIPBL. These results advance our understanding of cohesin by establishing direct experimental evidence for a multistep DNA-binding mechanism mediated by dynamic protein conformational changes.}, number={11}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Kaur, Parminder and Lu, Xiaotong and Xu, Qi and Irvin, Elizabeth Marie and Pappas, Colette and Zhang, Hongshan and Finkelstein, Ilya J. and Shi, Zhubing and Tao, Yizhi Jane and Yu, Hongtao and et al.}, year={2023}, month={Nov} }
 @article{laspata_kaur_mersaoui_muoio_liu_bannister_nguyen_curry_pascal_poirier_et al._2023, title={PARP1 associates with R-loops to promote their resolution and genome stability}, volume={2}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkad066}, abstractNote={Abstract PARP1 is a DNA-dependent ADP-Ribose transferase with ADP-ribosylation activity that is triggered by DNA breaks and non-B DNA structures to mediate their resolution. PARP1 was also recently identified as a component of the R-loop-associated protein-protein interaction network, suggesting a potential role for PARP1 in resolving this structure. R-loops are three-stranded nucleic acid structures that consist of a RNA–DNA hybrid and a displaced non-template DNA strand. R-loops are involved in crucial physiological processes but can also be a source of genome instability if persistently unresolved. In this study, we demonstrate that PARP1 binds R-loops in vitro and associates with R-loop formation sites in cells which activates its ADP-ribosylation activity. Conversely, PARP1 inhibition or genetic depletion causes an accumulation of unresolved R-loops which promotes genomic instability. Our study reveals that PARP1 is a novel sensor for R-loops and highlights that PARP1 is a suppressor of R-loop-associated genomic instability.}, journal={NUCLEIC ACIDS RESEARCH}, author={Laspata, Natalie and Kaur, Parminder and Mersaoui, Sofiane Yacine and Muoio, Daniela and Liu, Zhiyan Silvia and Bannister, Maxwell Henry and Nguyen, Hai Dang and Curry, Caroline and Pascal, John M. and Poirier, Guy G. and et al.}, year={2023}, month={Feb} }
 @article{irvin_wang_2023, title={Single-molecule imaging of genome maintenance proteins encountering specific DNA sequences and structures}, volume={128}, ISSN={["1568-7856"]}, DOI={10.1016/j.dnarep.2023.103528}, abstractNote={DNA repair pathways are tightly regulated processes that recognize specific hallmarks of DNA damage and coordinate lesion repair through discrete mechanisms, all within the context of a three-dimensional chromatin landscape. Dysregulation or malfunction of any one of the protein constituents in these pathways can contribute to aging and a variety of diseases. While the collective action of these many proteins is what drives DNA repair on the organismal scale, it is the interactions between individual proteins and DNA that facilitate each step of these pathways. In much the same way that ensemble biochemical techniques have characterized the various steps of DNA repair pathways, single-molecule imaging (SMI) approaches zoom in further, characterizing the individual protein-DNA interactions that compose each pathway step. SMI techniques offer the high resolving power needed to characterize the molecular structure and functional dynamics of individual biological interactions on the nanoscale. In this review, we highlight how our lab has used SMI techniques – traditional atomic force microscopy (AFM) imaging in air, high-speed AFM (HS-AFM) in liquids, and the DNA tightrope assay – over the past decade to study protein-nucleic acid interactions involved in DNA repair, mitochondrial DNA replication, and telomere maintenance. We discuss how DNA substrates containing specific DNA sequences or structures that emulate DNA repair intermediates or telomeres were generated and validated. For each highlighted project, we discuss novel findings made possible by the spatial and temporal resolution offered by these SMI techniques and unique DNA substrates.}, journal={DNA REPAIR}, author={Irvin, Elizabeth Marie and Wang, Hong}, year={2023}, month={Aug} }
 @article{wojtaszek_hoff_longley_kaur_andres_wang_williams_copeland_2023, title={Structure-specific roles for PolG2-DNA complexes in maintenance and replication of mitochondrial DNA}, volume={8}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkad679}, abstractNote={Abstract The homodimeric PolG2 accessory subunit of the mitochondrial DNA polymerase gamma (Pol γ) enhances DNA binding and processive DNA synthesis by the PolG catalytic subunit. PolG2 also directly binds DNA, although the underlying molecular basis and functional significance are unknown. Here, data from Atomic Force Microscopy (AFM) and X-ray structures of PolG2–DNA complexes define dimeric and hexameric PolG2 DNA binding modes. Targeted disruption of PolG2 DNA-binding interfaces impairs processive DNA synthesis without diminishing Pol γ subunit affinities. In addition, a structure-specific DNA-binding role for PolG2 oligomers is supported by X-ray structures and AFM showing that oligomeric PolG2 localizes to DNA crossings and targets forked DNA structures resembling the mitochondrial D-loop. Overall, data indicate that PolG2 DNA binding has both PolG-dependent and -independent functions in mitochondrial DNA replication and maintenance, which provide new insight into molecular defects associated with PolG2 disruption in mitochondrial disease.}, journal={NUCLEIC ACIDS RESEARCH}, author={Wojtaszek, Jessica L. and Hoff, Kirsten E. and Longley, Matthew J. and Kaur, Parminder and Andres, Sara N. and Wang, Hong and Williams, R. Scott and Copeland, William C.}, year={2023}, month={Aug} }
 @article{leighton_irvin_kaur_liu_you_bhattaram_piehler_riehn_wang_pan_et al._2022, title={Densely methylated DNA traps Methyl-CpG-binding domain protein 2 but permits free diffusion by Methyl-CpG-binding domain protein 3}, volume={298}, ISSN={["1083-351X"]}, url={https://doi.org/10.1016/j.jbc.2022.102428}, DOI={10.1016/j.jbc.2022.102428}, abstractNote={The methyl-CpG–binding domain 2 and 3 proteins (MBD2 and MBD3) provide structural and DNA-binding function for the Nucleosome Remodeling and Deacetylase (NuRD) complex. The two proteins form distinct NuRD complexes and show different binding affinity and selectivity for methylated DNA. Previous studies have shown that MBD2 binds with high affinity and selectivity for a single methylated CpG dinucleotide while MBD3 does not. However, the NuRD complex functions in regions of the genome that contain many CpG dinucleotides (CpG islands). Therefore, in this work, we investigate the binding and diffusion of MBD2 and MBD3 on more biologically relevant DNA templates that contain a large CpG island or limited CpG sites. Using a combination of single-molecule and biophysical analyses, we show that both MBD2 and MBD3 diffuse freely and rapidly across unmethylated CpG-rich DNA. In contrast, we found methylation of large CpG islands traps MBD2 leading to stable and apparently static binding on the CpG island while MBD3 continues to diffuse freely. In addition, we demonstrate both proteins bend DNA, which is augmented by methylation. Together, these studies support a model in which MBD2-NuRD strongly localizes to and compacts methylated CpG islands while MBD3-NuRD can freely mobilize nucleosomes independent of methylation status. The methyl-CpG–binding domain 2 and 3 proteins (MBD2 and MBD3) provide structural and DNA-binding function for the Nucleosome Remodeling and Deacetylase (NuRD) complex. The two proteins form distinct NuRD complexes and show different binding affinity and selectivity for methylated DNA. Previous studies have shown that MBD2 binds with high affinity and selectivity for a single methylated CpG dinucleotide while MBD3 does not. However, the NuRD complex functions in regions of the genome that contain many CpG dinucleotides (CpG islands). Therefore, in this work, we investigate the binding and diffusion of MBD2 and MBD3 on more biologically relevant DNA templates that contain a large CpG island or limited CpG sites. Using a combination of single-molecule and biophysical analyses, we show that both MBD2 and MBD3 diffuse freely and rapidly across unmethylated CpG-rich DNA. In contrast, we found methylation of large CpG islands traps MBD2 leading to stable and apparently static binding on the CpG island while MBD3 continues to diffuse freely. In addition, we demonstrate both proteins bend DNA, which is augmented by methylation. Together, these studies support a model in which MBD2-NuRD strongly localizes to and compacts methylated CpG islands while MBD3-NuRD can freely mobilize nucleosomes independent of methylation status. The methyl-CpG–binding domain (MBD) family of proteins binds methylated DNA through a conserved domain that recognizes the symmetrically related methylcytosines in a cytosine-guanosine dinucleotide (CpG) (1Hendrich B. Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins.Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1089) Google Scholar). The structure of this domain bound to a single methylated CpG (mCpG) site has been determined for most members of the MBD family (2Ohki I. Shimotake N. Fujita N. Jee J. Ikegami T. Nakao M. et al.Solution structure of the methyl-CpG binding domain of human MBD1 in complex with methylated DNA.Cell. 2001; 105: 487-497Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 3Ho K.L. McNae I.W. Schmiedeberg L. Klose R.J. Bird A.P. Walkinshaw M.D. MeCP2 binding to DNA depends upon hydration at methyl-CpG.Mol. Cell. 2008; 29: 525-531Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 4Scarsdale J.N. Webb H.D. Ginder G.D. Williams Jr., D.C. Solution structure and dynamic analysis of chicken MBD2 methyl binding domain bound to a target-methylated DNA sequence.Nucleic Acids Res. 2011; 39: 6741-6752Crossref PubMed Scopus (80) Google Scholar, 5Walavalkar N.M. Cramer J.M. Buchwald W.A. Scarsdale J.N. Williams Jr., D.C. Solution structure and intramolecular exchange of methyl-cytosine binding domain protein 4 (MBD4) on DNA suggests a mechanism to scan for mCpG/TpG mismatches.Nucleic Acids Res. 2014; 42: 11218-11232Crossref PubMed Scopus (24) Google Scholar, 6Cramer J.M. Scarsdale J.N. Walavalkar N.M. Buchwald W.A. Ginder G.D. Williams Jr., D.C. Probing the dynamic distribution of bound states for methylcytosine-binding domains on DNA.J. Biol. 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Bird A. CpG islands and the regulation of transcription.Genes Dev. 2011; 25: 1010-1022Crossref PubMed Scopus (2147) Google Scholar, 11Portela A. Liz J. Nogales V. Setien F. Villanueva A. Esteller M. DNA methylation determines nucleosome occupancy in the 5'-CpG islands of tumor suppressor genes.Oncogene. 2013; 32: 5421-5428Crossref PubMed Scopus (33) Google Scholar, 12Collings C.K. Anderson J.N. Links between DNA methylation and nucleosome occupancy in the human genome.Epigenetics Chromatin. 2017; 10: 18Crossref PubMed Scopus (41) Google Scholar, 13Collings C.K. Waddell P.J. Anderson J.N. Effects of DNA methylation on nucleosome stability.Nucleic Acids Res. 2013; 41: 2918-2931Crossref PubMed Scopus (68) Google Scholar, 14Doi A. Park I.H. Wen B. Murakami P. Aryee M.J. Irizarry R. et al.Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts.Nat. Genet. 2009; 41: 1350-1353Crossref PubMed Scopus (965) Google Scholar). Furthermore, methylation of CpG islands in promoters and enhancers correlates with nucleosome occupancy, chromatin compaction, and associated gene silencing. Hence, we have investigated how MBD proteins bind and diffuse along these CpG islands to better understand the functional consequences in a more biologically relevant context. In the current work, we focus on the structure and dynamics of the MBD2 and MBD3 proteins. These two highly homologous proteins arose from a duplication of the ancestral MBD present across the animal kingdom (1Hendrich B. Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins.Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1089) Google Scholar, 15Cramer J.M. Pohlmann D. Gomez F. Mark L. Kornegay B. Hall C. et al.Methylation specific targeting of a chromatin remodeling complex from sponges to humans.Sci. Rep. 2017; 740674Crossref Scopus (20) Google Scholar). They contribute to the structure and function of the Nucleosome Remodeling and Deacetylase (NuRD) (16Le Guezennec X. Vermeulen M. Brinkman A.B. Hoeijmakers W.A. Cohen A. Lasonder E. et al.MBD2/NuRD and MBD3/NuRD, two distinct complexes with different biochemical and functional properties.Mol. Cell. Biol. 2006; 26: 843-851Crossref PubMed Scopus (266) Google Scholar) complex that can reposition nucleosomes, deacetylate histones, and modify gene expression. The NuRD complex (17Torrado M. Low J.K.K. Silva A.P.G. Schmidberger J.W. Sana M. Sharifi Tabar M. et al.Refinement of the subunit interaction network within the nucleosome remodelling and deacetylase (NuRD) complex.FEBS J. 2017; 284: 4216-4232Crossref PubMed Scopus (39) Google Scholar, 18Allen H.F. Wade P.A. Kutateladze T.G. The NuRD architecture.Cell. Mol. Life Sci. 2013; 70: 3513-3524Crossref PubMed Scopus (126) Google Scholar, 19Zhang W. Aubert A. Gomez de Segura J.M. Karuppasamy M. Basu S. Murthy A.S. et al.The nucleosome remodeling and deacetylase complex NuRD is built from preformed catalytically active sub-modules.J. Mol. Biol. 2016; 428: 2931-2942Crossref PubMed Scopus (47) Google Scholar, 20Zhang Y. Ng H.H. Erdjument-Bromage H. Tempst P. Bird A. Reinberg D. Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation.Genes Dev. 1999; 13: 1924-1935Crossref PubMed Scopus (937) Google Scholar) consists of a least six additional proteins, each of which has multiple paralogs that provide histone deacetylase activity (HDAC1/2), histone binding, and chromatin remodeling function (CHD3/4), and protein–protein interactions (GATAD2A/B, RBBP4/7, MTA1/2/3, CDK2AP1). The MBD2 and MBD3 proteins form distinct NuRD complexes that appear to have unique functional roles (16Le Guezennec X. Vermeulen M. Brinkman A.B. Hoeijmakers W.A. Cohen A. Lasonder E. et al.MBD2/NuRD and MBD3/NuRD, two distinct complexes with different biochemical and functional properties.Mol. Cell. Biol. 2006; 26: 843-851Crossref PubMed Scopus (266) Google Scholar, 21Leighton G. Williams Jr., D.C. The methyl-CpG-binding domain 2 and 3 proteins and formation of the nucleosome remodeling and deacetylase complex.J. Mol. Biol. 2020; 432: 1624-1639Crossref Scopus (18) Google Scholar, 22Yu X. Azzo A. Bilinovich S.M. Li X. Dozmorov M. Kurita R. et al.Disruption of the MBD2-NuRD complex but not MBD3-NuRD induces high level HbF expression in human adult erythroid cells.Haematologica. 2019; 104: 2361-2371Crossref PubMed Scopus (32) Google Scholar). The two proteins show different levels of selectivity for mCpGs attributable primarily to a single amino acid change from tyrosine (MBD2) to phenylalanine (MBD3) within the DNA-binding site (Fig. 1) (1Hendrich B. Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins.Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1089) Google Scholar, 6Cramer J.M. Scarsdale J.N. Walavalkar N.M. Buchwald W.A. Ginder G.D. Williams Jr., D.C. Probing the dynamic distribution of bound states for methylcytosine-binding domains on DNA.J. Biol. Chem. 2014; 289: 1294-1302Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 23Baubec T. Ivanek R. Lienert F. Schubeler D. Methylation-dependent and -independent genomic targeting principles of the MBD protein family.Cell. 2013; 153: 480-492Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 24Saito M. Ishikawa F. The mCpG-binding domain of human MBD3 does not bind to mCpG but interacts with NuRD/Mi2 components HDAC1 and MTA2.J. Biol. Chem. 2002; 277: 35434-35439Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). MBD2 shows up to 100-fold selectivity for a fully mCpG dinucleotide compared to an unmethylated CpG (1Hendrich B. Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins.Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1089) Google Scholar, 4Scarsdale J.N. Webb H.D. Ginder G.D. Williams Jr., D.C. Solution structure and dynamic analysis of chicken MBD2 methyl binding domain bound to a target-methylated DNA sequence.Nucleic Acids Res. 2011; 39: 6741-6752Crossref PubMed Scopus (80) Google Scholar, 25Desai M.A. Webb H.D. Sinanan L.M. Scarsdale J.N. Walavalkar N.M. Ginder G.D. et al.An intrinsically disordered region of methyl-CpG binding domain protein 2 (MBD2) recruits the histone deacetylase core of the NuRD complex.Nucleic Acids Res. 2015; 43: 3100-3113Crossref PubMed Scopus (44) Google Scholar, 26Hashimoto H. Liu Y. Upadhyay A.K. Chang Y. Howerton S.B. Vertino P.M. et al.Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation.Nucleic Acids Res. 2012; 40: 4841-4849Crossref PubMed Scopus (356) Google Scholar). In contrast, MBD3 binds DNA with an overall much lower affinity and shows no or slight (3–5 fold) selectivity for an mCpG (6Cramer J.M. Scarsdale J.N. Walavalkar N.M. Buchwald W.A. Ginder G.D. Williams Jr., D.C. Probing the dynamic distribution of bound states for methylcytosine-binding domains on DNA.J. Biol. Chem. 2014; 289: 1294-1302Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 26Hashimoto H. Liu Y. Upadhyay A.K. Chang Y. Howerton S.B. Vertino P.M. et al.Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation.Nucleic Acids Res. 2012; 40: 4841-4849Crossref PubMed Scopus (356) Google Scholar). Consistent with this binding difference, previous genomic localization studies found that MBD2-NuRD predominantly binds at heavily mCpG islands associated with silenced genes, whereas MBD3 localizes to methylated and unmethylated CpG islands associated with expressed genes (23Baubec T. Ivanek R. Lienert F. Schubeler D. Methylation-dependent and -independent genomic targeting principles of the MBD protein family.Cell. 2013; 153: 480-492Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 27Menafra R. Brinkman A.B. Matarese F. Franci G. Bartels S.J. Nguyen L. et al.Genome-wide binding of MBD2 reveals strong preference for highly methylated loci.PLoS One. 2014; 9: e99603Crossref PubMed Scopus (37) Google Scholar, 28Shimbo T. Du Y. Grimm S.A. Dhasarathy A. Mav D. Shah R.R. et al.MBD3 localizes at promoters, gene bodies and enhancers of active genes.Plos Genet. 2013; 9e1004028Crossref PubMed Scopus (82) Google Scholar, 29Gunther K. Rust M. Leers J. Boettger T. Scharfe M. Jarek M. et al.Differential roles for MBD2 and MBD3 at methylated CpG islands, active promoters and binding to exon sequences.Nucleic Acids Res. 2013; 41: 3010-3021Crossref PubMed Scopus (90) Google Scholar). However, recent data suggests the alternative interpretation that both MBD2 and MBD3 depend on methylation for proper localization across the genome (30Hainer S.J. McCannell K.N. Yu J. Ee L.-S. Zhu L.J. Rando O.J. et al.DNA methylation directs genomic localization of Mbd2 and Mbd3 in embryonic stem cells.Elife. 2016; 5: e21964Crossref PubMed Scopus (21) Google Scholar). Therefore, how methylation selectivity of the MBD2 and MBD3 proteins impacts this localization and function remains an open question in the field. In previous work, we used single-molecule analyses to study the behavior of the isolated MBD from MBD2 on various DNA substrates (31Pan H. Bilinovich S.M. Kaur P. Riehn R. Wang H. Williams Jr., D.C. CpG and methylation-dependent DNA binding and dynamics of the methylcytosine binding domain 2 protein at the single-molecule level.Nucleic Acids Res. 2017; 45: 9164-9177Crossref PubMed Scopus (19) Google Scholar). Consistent with NMR and bulk biochemical studies, we found a remarkable difference in DNA bending and sliding of the MBD from MBD2 (MBD2MBD) on methylated and unmethylated DNA containing CpG islands. MBD2MBD is mostly restricted to the mCpG islands, while it freely diffuses across unmethylated CpG islands. Furthermore, we also uncovered a novel role for the intrinsically disordered region (IDR) of MBD2 in DNA bending (25Desai M.A. Webb H.D. Sinanan L.M. Scarsdale J.N. Walavalkar N.M. Ginder G.D. et al.An intrinsically disordered region of methyl-CpG binding domain protein 2 (MBD2) recruits the histone deacetylase core of the NuRD complex.Nucleic Acids Res. 2015; 43: 3100-3113Crossref PubMed Scopus (44) Google Scholar). The DNA-bending angle induced by both the MBD and a small portion of the IDR (MBD2MBD+IDR) on unmethylated CpG-rich DNA is larger than observed for CpG-free and further increases upon binding mCpG-rich DNA. In separate structural studies of MBD3, we found that the MBD from MBD3 shows only weak selectivity for a single mCpG within a small (17 bp) dsDNA fragment (6Cramer J.M. Scarsdale J.N. Walavalkar N.M. Buchwald W.A. Ginder G.D. Williams Jr., D.C. Probing the dynamic distribution of bound states for methylcytosine-binding domains on DNA.J. Biol. Chem. 2014; 289: 1294-1302Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Based on a combination of chemical shift analyses, mutagenesis, and residual dipolar coupling measurements, we showed that MBD3 exchanges rapidly between CpG-specific and nonspecific binding modes, leading to chemical shift averaging between these two states. Hence, MBD3 recognizes an mCpG site, as evidenced by significant chemical shift changes, but does not strongly localize to this site when bound to DNA. This difference between the DNA-binding dynamics of MBD2 and MBD3 correlates with the prior localization studies that show both MBD2 and MBD3 localize to unmethylated CpG islands, while MBD2 more exclusively localizes to mCpG islands (23Baubec T. Ivanek R. Lienert F. Schubeler D. Methylation-dependent and -independent genomic targeting principles of the MBD protein family.Cell. 2013; 153: 480-492Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 27Menafra R. Brinkman A.B. Matarese F. Franci G. Bartels S.J. Nguyen L. et al.Genome-wide binding of MBD2 reveals strong preference for highly methylated loci.PLoS One. 2014; 9: e99603Crossref PubMed Scopus (37) Google Scholar, 28Shimbo T. Du Y. Grimm S.A. Dhasarathy A. Mav D. Shah R.R. et al.MBD3 localizes at promoters, gene bodies and enhancers of active genes.Plos Genet. 2013; 9e1004028Crossref PubMed Scopus (82) Google Scholar, 29Gunther K. Rust M. Leers J. Boettger T. Scharfe M. Jarek M. et al.Differential roles for MBD2 and MBD3 at methylated CpG islands, active promoters and binding to exon sequences.Nucleic Acids Res. 2013; 41: 3010-3021Crossref PubMed Scopus (90) Google Scholar). Despite these recent studies, we do not know how the remaining, largely unstructured regions of MBD2 and MBD3 influence diffusion along DNA. Furthermore, it is unclear how reduced selectivity and binding affinity of MBD3 modifies its distribution and sliding on methylated and unmethylated CpG islands, which contain many CpG sites. To address these questions, in the current studies, we use a combination of biophysical techniques, including atomic force microscopy (AFM) imaging (32Benarroch-Popivker D. Pisano S. Mendez-Bermudez A. Lototska L. Kaur P. Bauwens S. et al.TRF2-Mediated control of telomere DNA topology as a mechanism for chromosome-end protection.Mol. Cell. 2016; 61: 274-286Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 33Wang H. Tessmer I. Croteau D.L. Erie D.A. Van Houten B. Functional characterization and atomic force microscopy of a DNA repair protein conjugated to a quantum dot.Nano Lett. 2008; 8: 1631-1637Crossref PubMed Scopus (52) Google Scholar, 34Wang H. Yang Y. Schofield M.J. Du C. Fridman Y. Lee S.D. et al.DNA bending and unbending by MutS govern mismatch recognition and specificity.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14822-14827Crossref PubMed Scopus (164) Google Scholar), DNA tightrope assays (35Lin J. Countryman P. Chen H. Pan H. Fan Y. Jiang Y. et al.Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing.Nucleic Acids Res. 2016; 44: 6363-6376Crossref PubMed Scopus (24) Google Scholar, 36Kad N.M. Wang H. Kennedy G.G. Warshaw D.M. Van Houten B. Collaborative dynamic DNA scanning by nucleotide excision repair proteins investigated by single- molecule imaging of quantum-dot-labeled proteins.Mol. Cell. 2010; 37: 702-713Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 37Lin J. Countryman P. Buncher N. Kaur P. E L. Zhang Y. et al.TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.Nucleic Acids Res. 2014; 42: 2493-2504Crossref PubMed Scopus (51) Google Scholar), and NMR (5Walavalkar N.M. Cramer J.M. Buchwald W.A. Scarsdale J.N. Williams Jr., D.C. Solution structure and intramolecular exchange of methyl-cytosine binding domain protein 4 (MBD4) on DNA suggests a mechanism to scan for mCpG/TpG mismatches.Nucleic Acids Res. 2014; 42: 11218-11232Crossref PubMed Scopus (24) Google Scholar, 6Cramer J.M. Scarsdale J.N. Walavalkar N.M. Buchwald W.A. Ginder G.D. Williams Jr., D.C. Probing the dynamic distribution of bound states for methylcytosine-binding domains on DNA.J. Biol. Chem. 2014; 289: 1294-1302Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) to measure the binding and sliding of MBD2 and MBD3 on methylated and unmethylated DNA substrates. In the DNA tightrope assay, DNA molecules are stretched under hydrodynamic flow inside a flow cell. Anchoring of stretched DNA between poly-L-lysine–coated silica microspheres leads to the formation of DNA tightropes at an elongation of ∼90% of the DNA contour length (Fig. 2A). The spatial resolution of the DNA tightrope assay was estimated to be 16 nm (37Lin J. Countryman P. Buncher N. Kaur P. E L. Zhang Y. et al.TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.Nucleic Acids Res. 2014; 42: 2493-2504Crossref PubMed Scopus (51) Google Scholar). Uniquely, DNA tightropes created using tandemly ligated DNA allow us to directly correlate DNA-binding events with the underlying specific DNA sequences or structures such as three-stranded R-loops (31Pan H. Bilinovich S.M. Kaur P. Riehn R. Wang H. Williams Jr., D.C. CpG and methylation-dependent DNA binding and dynamics of the methylcytosine binding domain 2 protein at the single-molecule level.Nucleic Acids Res. 2017; 45: 9164-9177Crossref PubMed Scopus (19) Google Scholar, 32Benarroch-Popivker D. Pisano S. Mendez-Bermudez A. Lototska L. Kaur P. Bauwens S. et al.TRF2-Mediated control of telomere DNA topology as a mechanism for chromosome-end protection.Mol. Cell. 2016; 61: 274-286Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 37Lin J. Countryman P. Buncher N. Kaur P. E L. Zhang Y. et al.TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.Nucleic Acids Res. 2014; 42: 2493-2504Crossref PubMed Scopus (51) Google Scholar, 38Pan H. Jin M. Ghadiyaram A. Kaur P. Miller H.E. Ta H.M. et al.Cohesin SA1 and SA2 are RNA binding proteins that localize to RNA containing regions on DNA.Nucleic Acids Res. 2020; 48: 5639-5655Crossref PubMed Google Scholar). To study MBD proteins diffusion, we ligated linear DNA fragments to form DNA tightropes with CpG free or alternating CpG-free and CpG-rich regions (Fig. 2B and S8A). The results from our previous study revealed that the isolated MBD from MBD2, with or without a small portion of the adjacent IDR (MBD2MBD and MBD2MBD+IDR), carry out unbiased 1D diffusion on CpG-rich DNA but undergoes subdiffusion on CpG-free DNA. In contrast, both proteins stably and statically bind to mCpG regions. In this study, we purified a construct that contains almost the entire length of the MBD2b isoform plus the coiled-coil region from GATAD2A (MBD2sc) to investigate how these additional regions impact DNA binding and diffusion (Experimental Procedures, Fig. 1A). We previously found that the MBD2sc binds DNA with an approximately 100× higher affinity than MBD2MBD (25Desai M.A. Webb H.D. Sinanan L.M. Scarsdale J.N. Walavalkar N.M. Ginder G.D. et al.An intrinsically disordered region of methyl-CpG binding domain protein 2 (MBD2) recruits the histone deacetylase core of the NuRD complex.Nucleic Acids Res. 2015; 43: 3100-3113Crossref PubMed Scopus (44) Google Scholar). Consistent with these results, fluorescence anisotropy experiments showed that MBD2sc binds to DNA containing unmethylated, methylated, or no CpG site with equilibrium dissociation constants of 2.8 (±0.1 μM), 0.007 (±0.002 μM), or 9.1 (±0.4 μM), respectively (Fig. 2C). Hence, we questioned whether the additional binding affinity provided by the IDR in MBD2 would modify DNA binding and sliding. We directly addressed this question using the DNA tightrope assay. For the DNA tightrope assay, we first generated DNA substrates by tandemly ligating linear DNA fragments containing CpG-free or unmethylated CpG-free–rich sequences (Fig. 2B). Further, we conjugated His-tagged MBD2sc to streptavidin-coated quantum dots (SAv-QDs) through the multivalent chelator tris-nitrilotriacetic acid linker (Fig. 2A) (39Reichel A. Schaible D. Al Furoukh N. Cohen M. Schreiber G. Piehler J. Noncovalent, site-specific biotinylation of histidine-tagged proteins.Anal. Chem. 2007; 79: 8590-8600Crossref PubMed Scopus (47) Google Scholar). Following the formation of the DNA tightropes, we introduced QD-labeled MBD2sc into the flow cell. Analysis of MBD2sc on DNA tightropes revealed two populations (Table 1): apparently immobile throughout data acquisition and mobile molecules (Fig. 2D). To exclude that this apparently immobile population reflects aggregation, we categorized particles as a single protein or cluster based on their individual QD blinking rate (Table S1) which shows that only a small fraction comprises multiple proteins. To obtain diffusion coefficients for mobile MBD2sc on DNA tightropes, we tracked the position of MBD2sc-QDs on DNA by Gaussian fitting to kymographs (particle position versus time plots) (36Kad N.M. Wang H. Kennedy G.G. Warshaw D.M. Van Houten B. Collaborative dynamic DNA scanning by nucleotide excision repair proteins investigated by single- molecule imaging of quantum-dot-labeled proteins.Mol. Cell. 2010; 37: 702-713Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 40Dunn A.R. Kad N.M. Nelson S.R. Warshaw D.M. Wallace S.S. Single Qdot-labeled glycosylase molecules use a wedge amino acid to probe for lesions while scanning along DNA.Nucleic Acids Res. 2011; 39: 7487-7498Crossref PubMed Scopus (98) Google Scholar). We obtained diffusion coefficients and alpha exponents by fitting the mean square displacement (MSD) versus time. An alpha exponent of 1 indicates an unbiased random walk, and a value less than 1 indicates subdiffusion (41Saxton M.J. Single-particle tracking: The distribution of diffusion coefficients.Biophys. J. 1997; 72: 1744-1753Abstract Full Text PDF PubMed Scopus (479) Google Scholar). The diffusion coefficients displayed by MBD2sc on the CpG-free DNA tightrope were significantly slower than those on DNA tightropes containing CpG sites (CpG-free–rich) (Fig. 2E). In addition, on the CpG-free–rich tightropes, MBD2sc displayed alpha exponents close to 1 (1.0 ± 0.2), indicating largely unbiased 1D diffusion on DNA (Fig. 2F, Table 2). As compared to CpG-free–rich DNA, the alpha exponents for MBD2sc on CpG-free DNA tightropes were slightly (p < 0.001) reduced (0.8 ± 0.2, Fig. 2F, Table 2). Overall, MBD2sc displayed slightly different diffusion ranges on CpG-free DNA tightropes compared to CpG-free–rich DNA tightropes containing multiple CpG sites (Fig. S3). In summary, MBD2sc shows more rapid and extensive 1D diffusion on CpG-free–rich DNA than CpG-free sequences.Table 1Fraction of statically bound MBD2sc and MBD3sc on unmethylated- and methylated-DNA tightropesDNAMBD2scMBD3scStatic binding (%)NStatic binding (%)NCpG-free–rich23 ± 614747 ± 7123CpG-free19 ± 633354 ± 8240mCpG-free–rich96 ± 324652 ± 10345mCpG-mini90 ± 149550 ± 8158The values represent mean ± SD from 2 to 3 experiments for each data set. Open table in a new tab Table 2Diffusion coefficient of MBD2sc and MBD3sc on different DNA substratesDNAMBD2scMBD3scD (μm2/s)α exponentND (μm2/s)α exponentNCpG-free0.04 ± 0.020.8 ± 0.2990.04 ± 0.030.8 ± 0.3199CpG-free–rich0.15 ± 0.051.0 ± 0.21000.04 ± 0.010.9 ± 0.2144mCpG-free–richNA.NA.0.09 ± 0.021.0 ± 0.2174mCpG-miniNA.NA.0.05 ± 0.020.9 ± 0.170The values represent mean ± SD from 2 to 4 experiments for each data set. Open table in a new tab The values represent mean ± SD from 2 to 3 experiments for each data set. The values represent mean ± SD from 2 to 4 experiments for each data set. To evaluate how DNA methylation affects the dynamics of MBD2sc on DNA, we imaged QD-labeled MBD2sc on the CpG-free–rich DNA tightropes after methylation. Linear DNA substrates were methylated before ligation using CpG Methyltransferase (M.SssI) with SAM as a cofactor (Experimental procedures). We confirmed methylation of the linear CpG-free–rich DNA substrate by digestion with the methylation-sensitive HpaII restriction endonuclease (Fig. S1). We then tandemly ligated the mCpG-free–rich (mCpG-free-rich, Fig. 3A) and used it to form DNA tightropes between silica beads. Compared to unmethylated CpG-free–rich and CpG-free DNA, the binding density of MBD2sc increased approximately 4× on mCpG-free–rich DNA tightropes (Fig. 3B, S4A). This result is consistent with the preferential binding of MBD2sc to mCpG sites, as supported by binding affinity measurements (Fig. 2C). Notably, while the majority of MBD2sc observed on unmethylated CpG-free–rich DNA tightropes was mobile (77%), on mCpG-free–rich tightropes, the majority of the protein bound was apparently immobile (96%) throughout data acquisition (Fig. 3B and Table 1). Furthermore, the distance between adjacent proteins on the mCpG-free–rich DNA tightropes is Gaussian distributed, with the peak centered at 2.3 (±0.3 μm) (Fig. 3C). This spacing matches the calculated distances between adjacent mCpG-rich regions on DNA tightropes (Fig. 3A), considering that they are stretched to ∼90% of their contour length. Taken together, fluorescence imaging of MBD2sc on DNA tightropes establishes that MBD2sc recognizes mCpG islands through stable and apparently static binding. To evaluate whether MBD2s}, number={10}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Leighton, Gage O. and Irvin, Elizabeth Marie and Kaur, Parminder and Liu, Ming and You, Changjiang and Bhattaram, Dhruv and Piehler, Jacob and Riehn, Robert and Wang, Hong and Pan, Hai and et al.}, year={2022}, month={Oct} }
 @article{li_kaur_lo_chopra_smith_wang_gao_2022, title={Structural and dynamic basis of DNA capture and translocation by mitochondrial Twinkle helicase}, volume={11}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkac1089}, abstractNote={Abstract Twinkle is a mitochondrial replicative helicase which can self-load onto and unwind mitochondrial DNA. Nearly 60 mutations on Twinkle have been linked to human mitochondrial diseases. Using cryo-electron microscopy (cryo-EM) and high-speed atomic force microscopy (HS-AFM), we obtained the atomic-resolution structure of a vertebrate Twinkle homolog with DNA and captured in real-time how Twinkle is self-loaded onto DNA. Our data highlight the important role of the non-catalytic N-terminal domain of Twinkle. The N-terminal domain directly contacts the C-terminal helicase domain, and the contact interface is a hotspot for disease-related mutations. Mutations at the interface destabilize Twinkle hexamer and reduce helicase activity. With HS-AFM, we observed that a highly dynamic Twinkle domain, which is likely to be the N-terminal domain, can protrude ∼5 nm to transiently capture nearby DNA and initialize Twinkle loading onto DNA. Moreover, structural analysis and subunit doping experiments suggest that Twinkle hydrolyzes ATP stochastically, which is distinct from related helicases from bacteriophages.}, journal={NUCLEIC ACIDS RESEARCH}, author={Li, Zhuo and Kaur, Parminder and Lo, Chen-Yu and Chopra, Neil and Smith, Jamie and Wang, Hong and Gao, Yang}, year={2022}, month={Nov} }
 @article{pan_kaur_barnes_detwiler_sanford_liu_xu_mahn_tang_hao_et al._2021, title={Structure, dynamics, and regulation of TRF1-TIN2-mediated trans- and cis-interactions on telomeric DNA}, volume={297}, ISSN={["1083-351X"]}, DOI={10.1016/j.jbc.2021.101080}, abstractNote={TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD+ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation.}, number={3}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Pan, Hai and Kaur, Parminder and Barnes, Ryan and Detwiler, Ariana C. and Sanford, Samantha Lynn and Liu, Ming and Xu, Pengning and Mahn, Chelsea and Tang, Qingyu and Hao, Pengyu and et al.}, year={2021}, month={Sep} }
 @article{pan_kaur_barnes_detwiler_sanford_liu_xu_mahn_tang_hao_et al._2021, title={Structure, dynamics, and regulation of TRF1-TIN2-mediated trans-and cis-interactions on telomeric DNA}, journal={Journal of Biological Chemistry}, publisher={Elsevier}, author={Pan, Hai and Kaur, Parminder and Barnes, Ryan and Detwiler, Ariana C and Sanford, Samantha Lynn and Liu, Ming and Xu, Pengning and Mahn, Chelsea and Tang, Qingyu and Hao, Pengyu and et al.}, year={2021}, pages={101080} }
 @article{kaur_barnes_pan_detwiler_liu_mahn_hall_messenger_you_piehler_et al._2021, title={TIN2 is an architectural protein that facilitates TRF2-mediated trans- and cis-interactions on telomeric DNA}, volume={49}, ISSN={["1362-4962"]}, url={https://doi.org/10.1093/nar/gkab1142}, DOI={10.1093/nar/gkab1142}, abstractNote={Abstract The telomere specific shelterin complex, which includes TRF1, TRF2, RAP1, TIN2, TPP1 and POT1, prevents spurious recognition of telomeres as double-strand DNA breaks and regulates telomerase and DNA repair activities at telomeres. TIN2 is a key component of the shelterin complex that directly interacts with TRF1, TRF2 and TPP1. In vivo, the large majority of TRF1 and TRF2 are in complex with TIN2 but without TPP1 and POT1. Since knockdown of TIN2 also removes TRF1 and TRF2 from telomeres, previous cell-based assays only provide information on downstream effects after the loss of TRF1/TRF2 and TIN2. Here, we investigated DNA structures promoted by TRF2–TIN2 using single-molecule imaging platforms, including tracking of compaction of long mouse telomeric DNA using fluorescence imaging, atomic force microscopy (AFM) imaging of protein–DNA structures, and monitoring of DNA–DNA and DNA–RNA bridging using the DNA tightrope assay. These techniques enabled us to uncover previously unknown unique activities of TIN2. TIN2S and TIN2L isoforms facilitate TRF2-mediated telomeric DNA compaction (cis-interactions), dsDNA–dsDNA, dsDNA–ssDNA and dsDNA–ssRNA bridging (trans-interactions). Furthermore, TIN2 facilitates TRF2-mediated T-loop formation. We propose a molecular model in which TIN2 functions as an architectural protein to promote TRF2-mediated trans and cis higher-order nucleic acid structures at telomeres.}, number={22}, journal={NUCLEIC ACIDS RESEARCH}, publisher={Oxford University Press (OUP)}, author={Kaur, Parminder and Barnes, Ryan and Pan, Hai and Detwiler, Ariana C. and Liu, Ming and Mahn, Chelsea and Hall, Jonathan and Messenger, Zach and You, Changjiang and Piehler, Jacob and et al.}, year={2021}, month={Dec}, pages={13000–13018} }
 @article{kaur_pan_longley_copeland_wang_2021, title={Using Atomic Force Microscopy to Study the Real Time Dynamics of DNA Unwinding by Mitochondrial Twinkle Helicase}, volume={11}, ISSN={["2331-8325"]}, DOI={10.21769/BioProtoc.4139}, abstractNote={Understanding the structure and dynamics of DNA-protein interactions during DNA replication is crucial for elucidating the origins of disorders arising from its dysfunction. In this study, we employed Atomic Force Microscopy as a single-molecule imaging tool to examine the mitochondrial DNA helicase Twinkle and its interactions with DNA. We used imaging in air and time-lapse imaging in liquids to observe the DNA binding and unwinding activities of Twinkle hexamers at the single-molecule level. These procedures helped us visualize Twinkle loading onto and unloading from the DNA in the open-ring conformation. Using traditional methods, it has been shown that Twinkle is capable of unwinding dsDNA up to 20-55 bps. We found that the addition of mitochondrial single-stranded DNA binding protein (mtSSB) facilitates a 5-fold increase in the DNA unwinding rate for the Twinkle helicase. The protocols developed in this study provide new platforms to examine DNA replication and to explore the mechanism driving DNA deletion and human diseases. Graphic abstract: Mitochondrial Twinkle Helicase Dynamics.}, number={17}, journal={BIO-PROTOCOL}, author={Kaur, Parminder and Pan, Hai and Longley, Matthew J. and Copeland, William C. and Wang, Hong}, year={2021}, month={Sep} }
 @article{pan_jin_ghadiyaram_kaur_miller_ta_liu_fan_mahn_gorthi_et al._2020, title={Cohesin SA1 and SA2 are RNA binding proteins that localize to RNA containing regions on DNA}, volume={48}, ISSN={["1362-4962"]}, url={http://dx.doi.org/10.1093/nar/gkaa284}, DOI={10.1093/nar/gkaa284}, abstractNote={Abstract Cohesin SA1 (STAG1) and SA2 (STAG2) are key components of the cohesin complex. Previous studies have highlighted the unique contributions by SA1 and SA2 to 3D chromatin organization, DNA replication fork progression, and DNA double-strand break (DSB) repair. Recently, we discovered that cohesin SA1 and SA2 are DNA binding proteins. Given the recently discovered link between SA2 and RNA-mediated biological pathways, we investigated whether or not SA1 and SA2 directly bind to RNA using a combination of bulk biochemical assays and single-molecule techniques, including atomic force microscopy (AFM) and the DNA tightrope assay. We discovered that both SA1 and SA2 bind to various RNA containing substrates, including ssRNA, dsRNA, RNA:DNA hybrids, and R-loops. Importantly, both SA1 and SA2 localize to regions on dsDNA that contain RNA. We directly compared the SA1/SA2 binding and R-loops sites extracted from Chromatin Immunoprecipitation sequencing (ChIP-seq) and DNA-RNA Immunoprecipitation sequencing (DRIP-Seq) data sets, respectively. This analysis revealed that SA1 and SA2 binding sites overlap significantly with R-loops. The majority of R-loop-localized SA1 and SA2 are also sites where other subunits of the cohesin complex bind. These results provide a new direction for future investigation of the diverse biological functions of SA1 and SA2.}, number={10}, journal={NUCLEIC ACIDS RESEARCH}, publisher={Oxford University Press (OUP)}, author={Pan, Hai and Jin, Miao and Ghadiyaram, Ashwin and Kaur, Parminder and Miller, Henry E. and Ta, Hai Minh and Liu, Ming and Fan, Yanlin and Mahn, Chelsea and Gorthi, Aparna and et al.}, year={2020}, month={Jun}, pages={5639–5655} }
 @article{pan_jin_ghadiyaram_kaur_miller_ta_liu_fan_mahn_gorthi_et al._2020, title={Cohesin SA1 and SA2 are RNA binding proteins that localize to RNA containing regions on DNA}, journal={Nucleic Acids Research}, author={Pan, Hai and Jin, Miao and Ghadiyaram, Ashwin and Kaur, Parminder and Miller, Henry E and Ta, Hai Minh and Liu, Ming and Fan, Yanlin and Mahn, Chelsea and Gorthi, Aparna and et al.}, year={2020} }
 @article{liu_movahed_dangi_pan_kaur_bilinovich_faison_leighton_wang_williams_et al._2020, title={DNA looping by two 5-methylcytosine-binding proteins quantified using nanofluidic devices}, volume={13}, ISSN={["1756-8935"]}, DOI={10.1186/s13072-020-00339-7}, abstractNote={Abstract Background MeCP2 and MBD2 are members of a family of proteins that possess a domain that selectively binds 5-methylcytosine in a CpG context. Members of the family interact with other proteins to modulate DNA packing. Stretching of DNA–protein complexes in nanofluidic channels with a cross-section of a few persistence lengths allows us to probe the degree of compaction by proteins. Results We demonstrate DNA compaction by MeCP2 while MBD2 does not affect DNA configuration. By using atomic force microscopy (AFM), we determined that the mechanism for compaction by MeCP2 is the formation of bridges between distant DNA stretches and the formation of loops. Conclusions Despite sharing a similar specific DNA-binding domain, the impact of full-length 5-methylcytosine-binding proteins can vary drastically between strong compaction of DNA and no discernable large-scale impact of protein binding. We demonstrate that ATTO 565-labeled MBD2 is a good candidate as a staining agent for epigenetic mapping.}, number={1}, journal={EPIGENETICS & CHROMATIN}, publisher={Springer}, author={Liu, Ming and Movahed, Saeid and Dangi, Saroj and Pan, Hai and Kaur, Parminder and Bilinovich, Stephanie M. and Faison, Edgar M. and Leighton, Gage O. and Wang, Hong and Williams, David C., Jr. and et al.}, year={2020}, month={Mar} }
 @article{kaur_longley_pan_wang_countryman_wang_copeland_2020, title={Single-molecule level structural dynamics of DNA unwinding by human mitochondrial Twinkle helicase}, volume={295}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.RA120.012795}, abstractNote={Knowledge of the molecular events in mitochondrial DNA (mtDNA) replication is crucial to understanding the origins of human disorders arising from mitochondrial dysfunction. Twinkle helicase is an essential component of mtDNA replication. Here, we employed atomic force microscopy imaging in air and liquids to visualize ring assembly, DNA binding, and unwinding activity of individual Twinkle hexamers at the single-molecule level. We observed that the Twinkle subunits self-assemble into hexamers and higher-order complexes that can switch between open and closed-ring configurations in the absence of DNA. Our analyses helped visualize Twinkle loading onto and unloading from DNA in an open-ringed configuration. They also revealed that closed-ring conformers bind and unwind several hundred base pairs of duplex DNA at an average rate of ∼240 bp/min. We found that the addition of mitochondrial single-stranded (ss) DNA–binding protein both influences the ways Twinkle loads onto defined DNA substrates and stabilizes the unwound ssDNA product, resulting in a ∼5-fold stimulation of the apparent DNA-unwinding rate. Mitochondrial ssDNA-binding protein also increased the estimated translocation processivity from 1750 to >9000 bp before helicase disassociation, suggesting that more than half of the mitochondrial genome could be unwound by Twinkle during a single DNA-binding event. The strategies used in this work provide a new platform to examine Twinkle disease variants and the core mtDNA replication machinery. They also offer an enhanced framework to investigate molecular mechanisms underlying deletion and depletion of the mitochondrial genome as observed in mitochondrial diseases. Knowledge of the molecular events in mitochondrial DNA (mtDNA) replication is crucial to understanding the origins of human disorders arising from mitochondrial dysfunction. Twinkle helicase is an essential component of mtDNA replication. Here, we employed atomic force microscopy imaging in air and liquids to visualize ring assembly, DNA binding, and unwinding activity of individual Twinkle hexamers at the single-molecule level. We observed that the Twinkle subunits self-assemble into hexamers and higher-order complexes that can switch between open and closed-ring configurations in the absence of DNA. Our analyses helped visualize Twinkle loading onto and unloading from DNA in an open-ringed configuration. They also revealed that closed-ring conformers bind and unwind several hundred base pairs of duplex DNA at an average rate of ∼240 bp/min. We found that the addition of mitochondrial single-stranded (ss) DNA–binding protein both influences the ways Twinkle loads onto defined DNA substrates and stabilizes the unwound ssDNA product, resulting in a ∼5-fold stimulation of the apparent DNA-unwinding rate. Mitochondrial ssDNA-binding protein also increased the estimated translocation processivity from 1750 to >9000 bp before helicase disassociation, suggesting that more than half of the mitochondrial genome could be unwound by Twinkle during a single DNA-binding event. The strategies used in this work provide a new platform to examine Twinkle disease variants and the core mtDNA replication machinery. They also offer an enhanced framework to investigate molecular mechanisms underlying deletion and depletion of the mitochondrial genome as observed in mitochondrial diseases.}, number={17}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Kaur, Parminder and Longley, Matthew J. and Pan, Hai and Wang, Wendy and Countryman, Preston and Wang, Hong and Copeland, William C.}, year={2020}, month={Apr}, pages={5564–5576} }
 @article{kaur_longley_pan_wang_countryman_wang_copeland_2020, title={Single-molecule level structural dynamics of DNA unwinding by human mitochondrial Twinkle helicase}, volume={295}, number={17}, journal={Journal of Biological Chemistry}, publisher={ASBMB}, author={Kaur, Parminder and Longley, Matthew J and Pan, Hai and Wang, Wendy and Countryman, Preston and Wang, Hong and Copeland, William C}, year={2020}, pages={5564–5576} }
 @article{wang_ghadiyaram_pan_fan_kaur_gorthi_riehn_bishop_jane tao_2019, title={Cohesin SA2 and EWSR1 in R-Loop Regulation}, volume={116}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2018.11.2723}, DOI={10.1016/J.BPJ.2018.11.2723}, abstractNote={R-loops are three-stranded nucleic acid structures consisting of an RNA-DNA hybrid and a displaced single-stranded DNA (ssDNA) loop. R-loops collectively occupy up to 5% of the mammalian genome, which occur at conserved hotspot including promoter and terminator regions of poly(A) dependent genes. R-loops are proposed to be the “double-edged sword” that functions as powerful regulators of gene expression and induces genome instability if it is un-regulated. Despite the importance of R-loops in a wide range of biological pathways, proteins that mediate R-loop dependent cellular function are not fully understood. Recently The Bishop group showed that Ewing sarcoma cells displayed a higher level of R-loops compared to control cell lines. EWSR1 depletion induces R-loop accumulation. In addition, the second most common mutation in Ewing sarcoma cells was found in cohesin SA2. These observations raise the important question of whether EWSR1 as well as cohesin SA1 and SA2 subunits are sensors of R-loops that regulate R-loop processing. Using atomic force microscopy (AFM) and bulk fluorescence anisotropy, we tested the hypothesis that cohesin SA1, SA2, and EWSR1 directly bind to R-loops. We observed that cohesin SA1/SA2, and EWSR1 specifically recognize R-loops. These observations suggest direct roles of cohesin SA1/SA2 and EWSR1 in R-loop regulation and how the loss of SA2 in Ewing sarcoma may provide some benefit to these cancers.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Wang, Hong and Ghadiyaram, Ashwin and Pan, Hai and Fan, Yanlin and Kaur, Parminder and Gorthi, Aparna and Riehn, Robert and Bishop, Alexander J.R. and Jane Tao, Yizhi}, year={2019}, month={Feb}, pages={505a} }
 @article{fouquerel_barnes_wang_opresko_2019, title={Measuring UV Photoproduct Repair in Isolated Telomeres and Bulk Genomic DNA}, volume={1999}, ISBN={["978-1-4939-9499-1"]}, ISSN={["1940-6029"]}, DOI={10.1007/978-1-4939-9500-4_20}, abstractNote={Telomere repeats at chromosomal ends are essential for genome stability and sustained cellular proliferation but are susceptible to DNA damage. Repair of damage at telomeres is influenced by numerous factors including telomeric binding proteins, sequence and structure. Ultraviolet (UV) light irradiation induces DNA photoproducts at telomeres that can interfere with telomere maintenance. Here we describe a highly sensitive method for quantifying the formation and removal of UV photoproducts in telomeres isolated from UV irradiated cultured human cells. Damage is detected by immunospot blotting of telomeres with highly specific antibodies against UV photoproducts. This method is adaptable for measuring other types of DNA damage at telomeres as well.}, journal={DNA REPAIR: METHODS AND PROTOCOLS}, author={Fouquerel, Elise and Barnes, Ryan P. and Wang, Hong and Opresko, Patricia L.}, year={2019}, pages={295–306} }
 @inbook{fouquerel_barnes_wang_opresko_2019, title={Measuring UV Photoproduct Repair in Isolated Telomeres and Bulk Genomic DNA}, booktitle={DNA Repair}, publisher={Springer}, author={Fouquerel, Elise and Barnes, Ryan P and Wang, Hong and Opresko, Patricia L}, year={2019}, pages={295–306} }
 @article{kaur_barnes_pan_opresko_riehn_wang_2019, title={Single-Molecule Study of TRF2 Mediated DNA Compaction using Physiologically Relevant Long Telomeric DNA}, volume={116}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2018.11.2725}, DOI={10.1016/J.BPJ.2018.11.2725}, abstractNote={Telomeres are nucleoprotein structures that prevent the degradation or fusion of the ends of linear chromosomes by shielding them from activating DNA damage response (DDR) and DNA double-strand break (DSB) repair. Human telomeres contain ∼2 to 20 kb of TTAGGG repeats and a G-rich 3’ overhang. A specialized six-protein shelterin complex, including TRF1, TRF2, RAP1, TIN2, TPP1 and POT1, binds and prevents telomeres from being falsely recognized as double strand breaks, and regulates telomerase and DNA repair protein access. Extensive telomere shortening or dramatic telomere loss due to DNA damage causes chromosome ends to be recognized as DNA breaks, which triggers cell senescence and aging-related pathologies. Human telomeric DNA is arranged into T-loops, in which the 3’ single-stranded overhang invades the upstream double-stranded region. A recent study suggests that T-loops at telomeres function as conformational switches that regulate ATM activation [1]. Further, it was also shown that TRF2 mediated DNA compaction drives T-loop formation [2]. While it has been shown that TRF2 protein is required for the T-loop formation, essential information regarding the dynamics of its formation still remains unknown. Moreover, most of the previous studies were done using short telomeric DNA sequences ∼ 20 kb. To elucidate the dynamics of TRF2 mediated DNA compaction and T-loop formation, we used single molecule methods which frequently require long DNA substrates. We established a new method for extracting and purifying long telomeric DNA from mouse liver cells using Region specific Extraction (RSE) [3].}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Kaur, Parminder and Barnes, Ryan and Pan, Hai and Opresko, Patricia and Riehn, Robert and Wang, Hong}, year={2019}, month={Feb}, pages={505a} }
 @article{pan_dangi_kaur_hao_weninger_riehn_opresko_wang_2019, title={TIN2 is an Architectural Protein Stabilizing TRF1 at Telomere}, volume={116}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2018.11.1168}, DOI={10.1016/J.BPJ.2018.11.1168}, abstractNote={Telomeres, consisting of duplex TTAGGG repeats and associating with protein complexes at chromosome ends, play a crucial role in maintaining the stability of chromosomes. The protein complex - shelterin contains six subunits (TRF1, TRF2, RAP1, TIN2, TPP1 and POT1), which bind to telomeres and protect the chromosome ends from DNA repairing, and recruit telomerase when chromosome gets shortened abnormally. Among these six subunits, TIN2 has no affinity to either double-strand or single-strand DNA. However, it is a core component bridging the double-strand DNA binding proteins (TRF1 and TRF2) to single-strand DNA binding protein complex (TPP1-POT1). Loss of TRF1 or TRF2 binding domain in TIN2 can trigger DNA damage response. Moreover, TIN2 without the TPP1 binding domain is capable of fully supporting the stabilization of TRF1 and TRF2/RAP1. Despite the significance of TIN2 in telomere maintenance, the mechanism underlying TIN2 remains elusive. To understand how TIN2 affects TRF1 binding dynamics and how TIN2 influences with TRF1-mediated telomeric DNA pairing, we conducted Atomic Force Microscopy (AFM) and used nanochannels confined DNA to study the telomeric DNA conformation upon TRF1 and TIN2 binding. We observed high-order protein-DNA complexes formation indicating TIN2 facilitates TRF1 accumulation on telomeric DNA. We also carried out single molecule fluorescence technique to investigate how TIN2 affects TRF1 binding dynamics on telomeric DNA sequences. Our results show that TIN2 can accelerate DNA-DNA pairing and stabilize TRF1 on telomeric DNA.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Pan, Hai and Dangi, Saroj and Kaur, Parminder and Hao, Pengyu and Weninger, Keith and Riehn, Robert and Opresko, Patricia and Wang, Hong}, year={2019}, month={Feb}, pages={211a–212a} }
 @article{countryman_fan_gorthi_pan_strickland_kaur_wang_lin_lei_white_et al._2018, title={Cohesin SA2 is a sequence-independent DNA-binding protein that recognizes DNA replication and repair intermediates}, volume={293}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.m117.806406}, abstractNote={Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids, mediated by the cohesin protein complex, which also plays crucial roles in diverse genome maintenance pathways. Current models attribute DNA binding by cohesin to entrapment of dsDNA by the cohesin ring subunits (SMC1, SMC3, and RAD21 in humans). However, the biophysical properties and activities of the fourth core cohesin subunit SA2 (STAG2) are largely unknown. Here, using single-molecule atomic force and fluorescence microscopy imaging as well as fluorescence anisotropy measurements, we established that SA2 binds to both dsDNA and ssDNA, albeit with a higher binding affinity for ssDNA. We observed that SA2 can switch between the 1D diffusing (search) mode on dsDNA and stable binding (recognition) mode at ssDNA gaps. Although SA2 does not specifically bind to centromeric or telomeric sequences, it does recognize DNA structures often associated with DNA replication and double-strand break repair, such as a double-stranded end, single-stranded overhang, flap, fork, and ssDNA gap. SA2 loss leads to a defect in homologous recombination-mediated DNA double-strand break repair. These results suggest that SA2 functions at intermediate DNA structures during DNA transactions in genome maintenance pathways. These findings have important implications for understanding the function of cohesin in these pathways.}, number={3}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Countryman, Preston and Fan, Yanlin and Gorthi, Aparna and Pan, Hai and Strickland, Jack and Kaur, Parminder and Wang, Xuechun and Lin, Jiangguo and Lei, Xiaoying and White, Christian and et al.}, year={2018}, month={Jan}, pages={1054–1069} }
 @article{kaur_longley_pan_wang_copeland_2018, title={Single-molecule DREEM imaging reveals DNA wrapping around human mitochondrial single-stranded DNA binding protein}, volume={46}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gky875}, abstractNote={Improper maintenance of the mitochondrial genome progressively disrupts cellular respiration and causes severe metabolic disorders commonly termed mitochondrial diseases. Mitochondrial single-stranded DNA binding protein (mtSSB) is an essential component of the mtDNA replication machinery. We utilized single-molecule methods to examine the modes by which human mtSSB binds DNA to help define protein interactions at the mtDNA replication fork. Direct visualization of individual mtSSB molecules by atomic force microscopy (AFM) revealed a random distribution of mtSSB tetramers bound to extended regions of single-stranded DNA (ssDNA), strongly suggesting non-cooperative binding by mtSSB. Selective binding to ssDNA was confirmed by AFM imaging of individual mtSSB tetramers bound to gapped plasmid DNA substrates bearing defined single-stranded regions. Shortening of the contour length of gapped DNA upon binding mtSSB was attributed to DNA wrapping around mtSSB. Tracing the DNA path in mtSSB-ssDNA complexes with Dual-Resonance-frequency-Enhanced Electrostatic force Microscopy established a predominant binding mode with one DNA strand winding once around each mtSSB tetramer at physiological salt conditions. Single-molecule imaging suggests mtSSB may not saturate or fully protect single-stranded replication intermediates during mtDNA synthesis, leaving the mitochondrial genome vulnerable to chemical mutagenesis, deletions driven by primer relocation or other actions consistent with clinically observed deletion biases.}, number={21}, journal={NUCLEIC ACIDS RESEARCH}, author={Kaur, Parminder and Longley, Matthew J. and Pan, Hai and Wang, Hong and Copeland, William C.}, year={2018}, month={Nov}, pages={11287–11302} }
 @article{countryman_fan_gorthi_pan_strickland_kaur_wang_lin_lei_white_et al._2017, title={Cohesin SA2 is a sequence independent DNA binding protein that recognizes DNA replication and repair intermediates}, journal={Journal of Biological Chemistry}, publisher={ASBMB}, author={Countryman, Preston and Fan, Yanlin and Gorthi, Aparna and Pan, Hai and Strickland, Jack and Kaur, Parminder and Wang, Xuechun and Lin, Jianggguo and Lei, Xiaoying and White, Christian and et al.}, year={2017}, pages={jbc-M117} }
 @article{pan_bilinovich_kaur_riehn_wang_williams_2017, title={CpG and methylation-dependent DNA binding and dynamics of the methylcytosine binding domain 2 protein at the single-molecule level}, volume={45}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkx548}, abstractNote={The methylcytosine-binding domain 2 (MBD2) protein recruits the nucleosome remodeling and deacetylase complex (NuRD) to methylated DNA to modify chromatin and regulate transcription. Importantly, MBD2 functions within CpG islands that contain 100s to 1000s of potential binding sites. Since NuRD physically rearranges nucleosomes, the dynamic mobility of this complex is directly related to function. In these studies, we use NMR and single-molecule atomic force microscopy and fluorescence imaging to study DNA binding dynamics of MBD2. Single-molecule fluorescence tracking on DNA tightropes containing regions with CpG-rich and CpG-free regions reveals that MBD2 carries out unbiased 1D diffusion on CpG-rich DNA but subdiffusion on CpG-free DNA. In contrast, the protein stably and statically binds to methylated CpG (mCpG) regions. The intrinsically disordered region (IDR) on MBD2 both reduces exchange between mCpG sites along the DNA as well as the dissociation from DNA, acting like an anchor that restricts the dynamic mobility of the MBD domain. Unexpectedly, MBD2 binding to methylated CpGs induces DNA bending that is augmented by the IDR region of the protein. These results suggest that MBD2 targets NuRD to unmethylated or methylated CpG islands where its distinct dynamic binding modes help maintain open or closed chromatin, respectively.}, number={15}, journal={NUCLEIC ACIDS RESEARCH}, author={Pan, Hai and Bilinovich, Stephanie M. and Kaur, Parminder and Riehn, Robert and Wang, Hong and Williams, David C., Jr.}, year={2017}, month={Sep}, pages={9164–9177} }
 @article{adkins_swygert_kaur_niu_grigoryev_sung_wang_peterson_2017, title={Nucleosome-like, Single-stranded DNA (ssDNA)-Histone Octamer Complexes and the Implication for DNA Double Strand Break Repair}, volume={292}, number={13}, journal={Journal of Biological Chemistry}, publisher={ASBMB}, author={Adkins, Nicholas L and Swygert, Sarah G and Kaur, Parminder and Niu, Hengyao and Grigoryev, Sergei A and Sung, Patrick and Wang, Hong and Peterson, Craig L}, year={2017}, pages={5271–5281} }
 @article{erie_bradford_wilkins_bower_wang_gauer_satusky_qiu_weninger_kaur_et al._2017, title={Single Molecule Fluorescence and Atomic Force Microscopy Studies of DNA Repair}, volume={112}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2016.11.061}, DOI={10.1016/J.BPJ.2016.11.061}, abstractNote={DNA polymerases that are responsible for replication make approximately one error for every 107 bases copied, but the human genome contains ∼6 billion bases, which results in ∼600 errors per round of replication. The DNA mismatch repair (MMR) system corrects these DNA synthesis errors that occur during replication. MMR is initiated by the highly conserved MutS and MutL homologs, which are both dimers and contain DNA binding and ATPase activities that are essential for MMR in vivo. MutS homologs initiate repair by binding to a mismatch and undergoing an ATP-dependent conformational change that promotes its interaction with MutL homologs. This complex signals the initiation of excision and resynthesis of the newly synthesized DNA strand containing the incorrect nucleotide. We have been using a combination of atomic force microscopy (AFM) and single molecule fluorescence to characterize the stoichiometries and the conformational and dynamic properties of MutS and MutL homologs and their assembly on DNA containing a mismatch. We have also developed a new dual resonance frequency enhanced electrostatic force microscopy (DREEM), in which we simultaneously collect the AFM topographic image and an image of the electrostatic potential of the surface. The DREEM images reveal the path of DNA inside individual protein-DNA complexes, yielding unprecedented details about DNA conformations within simple and complicated complexes. I will discuss our studies on the assembly of MutS and MutL homologs on mismatches, with a focus on how AFM, DREEM, and single-molecule fluorescence can be powerful tools to study the stoichiometries, conformations, and dynamic assembly of multi-component complexes.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Erie, Dorothy and Bradford, Kira and Wilkins, Hunter and Bower, Jacqueline and Wang, Zimeng and Gauer, Jacob and Satusky, Matthew and Qiu, Rouyi and Weninger, Keith and Kaur, Parminder and et al.}, year={2017}, month={Feb}, pages={7a} }
 @article{leblanc_wilkins_li_kaur_wang_erie_2017, title={Using Atomic Force Microscopy to Characterize the Conformational Properties of Proteins and Protein--DNA Complexes That Carry Out DNA Repair}, volume={592}, journal={Methods in Enzymology}, publisher={Elsevier}, author={LeBlanc, Sharonda and Wilkins, Hunter and Li, Zimeng and Kaur, Parminder and Wang, Hong and Erie, Dorothy A}, year={2017}, pages={187–212} }
 @article{bilinovich_pan_kaur_wang_williams_2017, title={Walking the DNA Methylation Tightrope: The Involvement of Intrinsically Disordered Regions of Transcription Factors}, volume={112}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2016.11.1144}, DOI={10.1016/J.BPJ.2016.11.1144}, abstractNote={Most 5-methylcytosine-binding domain proteins (such as MeCP2 and MBD1-6) contain a highly conserved 70 amino acid methyl-CpG-binding domain (MBD). The founding member of vertebrate MBD proteins, MBD2, is associated with the Nucleosome Remodeling Deacetylase complex (NuRD) and exhibits DNA binding specificity for methylated CpG (mCpG) islands. MBD2 consists of the MBD, an intrinsically disorder region (IDR), and a coiled-coil region. The inclusion of the IDR in MBD2 increases binding affinity for mCpG. However, how MBD2 recognizes methylated DNA and the role of the IDR in influencing binding are not fully understood. To further elucidate the role of the IDR in DNA binding, we incorporated a series of biophysical techniques to examine movement of MBD2 without the IDR (MBD2MBD) or with a portion of the IDR (MBD2MBD+IDR). NMR and ZZ-exchange spectroscopy showed differences in the distribution and intramolecular exchange rate of the two protein constructs on 17bp dsDNA substrates that contain either CpG islands, mCpG sites, or no CpG sites. Single-molecule fluorescence tracking of quantum dot-labeled MBD2MBD and MBD2MBD+IDR on DNA tightropes containing CpG or mCpG reveals that both MBD2MBD and MBD2MBD+IDR search on DNA through 1D free diffusion and stably bind to methylated DNA. In conclusion, our data reveals how MBD2 dynamically achieves DNA binding specificity for methylated DNA and supports that the IDR of MBD2 modulates MBD2-binding dynamics. These results are important for further deciphering the molecular mechanisms of MBD and mCpG epigenetic regulation.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Bilinovich, Stephanie M. and Pan, Hai and Kaur, Parminder and Wang, Hong and Williams, David C., Jr}, year={2017}, month={Feb}, pages={207a} }
 @article{kaur_wu_lin_countryman_bradford_erie_riehn_opresko_wang_2016, title={Enhanced electrostatic force microscopy reveals higher-order DNA looping mediated by the telomeric protein TRF2}, volume={6}, ISSN={2045-2322}, url={http://dx.doi.org/10.1038/SREP20513}, DOI={10.1038/SREP20513}, abstractNote={Abstract Shelterin protein TRF2 modulates telomere structures by promoting dsDNA compaction and T-loop formation. Advancement of our understanding of the mechanism underlying TRF2-mediated DNA compaction requires additional information regarding DNA paths in TRF2-DNA complexes. To uncover the location of DNA inside protein-DNA complexes, we recently developed the D ual- R esonance-frequency- E nhanced E lectrostatic force M icroscopy (DREEM) imaging technique. DREEM imaging shows that in contrast to chromatin with DNA wrapping around histones, large TRF2-DNA complexes (with volumes larger than TRF2 tetramers) compact DNA inside TRF2 with portions of folded DNA appearing at the edge of these complexes. Supporting coarse-grained molecular dynamics simulations uncover the structural requirement and sequential steps during TRF2-mediated DNA compaction and result in folded DNA structures with protruding DNA loops as seen in DREEM imaging. Revealing DNA paths in TRF2 complexes provides new mechanistic insights into structure-function relationships underlying telomere maintenance pathways.}, number={1}, journal={Scientific Reports}, publisher={Springer Science and Business Media LLC}, author={Kaur, Parminder and Wu, Dong and Lin, Jiangguo and Countryman, Preston and Bradford, Kira C. and Erie, Dorothy A. and Riehn, Robert and Opresko, Patricia L. and Wang, Hong}, year={2016}, month={Feb} }
 @article{lin_countryman_chen_pan_fan_jiang_kaur_miao_gurgel_you_et al._2016, title={Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA--DNA pairing}, journal={Nucleic acids research}, publisher={Oxford Univ Press}, author={Lin, Jiangguo and Countryman, Preston and Chen, Haijiang and Pan, Hai and Fan, Yanlin and Jiang, Yunyun and Kaur, Parminder and Miao, Wang and Gurgel, Gisele and You, Changjiang and et al.}, year={2016}, pages={gkw518} }
 @article{lin_countryman_chen_pan_fan_jiang_kaur_miao_gurgel_you_et al._2016, title={Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing}, volume={44}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkw518}, abstractNote={Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids. Cohesion is thought to occur through the entrapment of DNA within the tripartite ring (Smc1, Smc3 and Rad21) with enforcement from a fourth subunit (SA1/SA2). Surprisingly, cohesin rings do not play a major role in sister telomere cohesion. Instead, this role is replaced by SA1 and telomere binding proteins (TRF1 and TIN2). Neither the DNA binding property of SA1 nor this unique telomere cohesion mechanism is understood. Here, using single-molecule fluorescence imaging, we discover that SA1 displays two-state binding on DNA: searching by one-dimensional (1D) free diffusion versus recognition through subdiffusive sliding at telomeric regions. The AT-hook motif in SA1 plays dual roles in modulating non-specific DNA binding and subdiffusive dynamics over telomeric regions. TRF1 tethers SA1 within telomeric regions that SA1 transiently interacts with. SA1 and TRF1 together form longer DNA–DNA pairing tracts than with TRF1 alone, as revealed by atomic force microscopy imaging. These results suggest that at telomeres cohesion relies on the molecular interplay between TRF1 and SA1 to promote DNA–DNA pairing, while along chromosomal arms the core cohesin assembly might also depend on SA1 1D diffusion on DNA and sequence-specific DNA binding.}, number={13}, journal={NUCLEIC ACIDS RESEARCH}, author={Lin, Jiangguo and Countryman, Preston and Chen, Haijiang and Pan, Hai and Fan, Yanlin and Jiang, Yunyun and Kaur, Parminder and Miao, Wang and Gurgel, Gisele and You, Changjiang and et al.}, year={2016}, month={Jul}, pages={6363–6376} }
 @article{benarroch-popivker_pisano_mendez-bermudez_lototska_kaur_bauwens_djerbi_latrick_fraisier_pei_et al._2016, title={TRF2-Mediated Control of Telomere DNA Topology as a Mechanism for Chromosome-End Protection}, journal={Molecular Cell}, publisher={Elsevier}, author={Benarroch-Popivker, Delphine and Pisano, Sabrina and Mendez-Bermudez, Aaron and Lototska, Liudmyla and Kaur, Parminder and Bauwens, Serge and Djerbi, Nadir and Latrick, Chrysa M and Fraisier, Vincent and Pei, Bei and et al.}, year={2016} }
 @article{benarroch-popivker_pisano_mendez-bermudez_lototska_kaur_bauwens_djerbi_latrick_fraisier_pei_et al._2016, title={TRF2-mediated control of telomere DNA topology as a mechanism for chromosome-end protection}, volume={61}, number={2}, journal={Molecular Cell}, author={Benarroch-Popivker, D. and Pisano, S. and Mendez-Bermudez, A. and Lototska, L. and Kaur, P. and Bauwens, S. and Djerbi, N. and Latrick, C. M. and Fraisier, V. and Pei, B. and et al.}, year={2016}, pages={274–286} }
 @article{wu_kaur_li_bradford_wang_erie_2016, title={Visualizing the Path of DNA through Proteins Using DREEM Imaging}, volume={61}, ISSN={["1097-4164"]}, DOI={10.1016/j.molcel.2015.12.012}, abstractNote={Many cellular functions require the assembly of multiprotein-DNA complexes. A growing area of structural biology aims to characterize these dynamic structures by combining atomic-resolution crystal structures with lower-resolution data from techniques that provide distributions of species, such as small-angle X-ray scattering, electron microscopy, and atomic force microscopy (AFM). A significant limitation in these combinatorial methods is localization of the DNA within the multiprotein complex. Here, we combine AFM with an electrostatic force microscopy (EFM) method to develop an exquisitely sensitive dual-resonance-frequency-enhanced EFM (DREEM) capable of resolving DNA within protein-DNA complexes. Imaging of nucleosomes and DNA mismatch repair complexes demonstrates that DREEM can reveal both the path of the DNA wrapping around histones and the path of DNA as it passes through both single proteins and multiprotein complexes. Finally, DREEM imaging requires only minor modifications of many existing commercial AFMs, making the technique readily available.}, number={2}, journal={MOLECULAR CELL}, publisher={Elsevier}, author={Wu, Dong and Kaur, Parminder and Li, Zimeng M. and Bradford, Kira C. and Wang, Hong and Erie, Dorothy A.}, year={2016}, month={Jan}, pages={315–323} }
 @article{countryman_lin_kaur_brennan_chen_you_piehler_tao_wang_2015, title={Determining the DNA Diffusion Behavior of SA2 on Various DNA Substrates}, volume={108}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2014.11.2177}, DOI={10.1016/J.BPJ.2014.11.2177}, abstractNote={Cohesin is a multi-protein complex involved in sister chromatid cohesion during cell replication and double strand DNA break repair. Cohesin core complex consists of a ring-like trimer and either SA1 or SA2 in somatic vertebrate cells. While SA1 and SA2 share ∼70% homology, only SA1 contains a critical AT hook domain responsible for its binding to telomere sequences. Cohesin-SA1 holds sister chromatids together at telomere regions during cell separation and can be found at specific promoter regions, while Cohesin-SA2 is predominantly located at intergenic and centromere regions. The mechanism by which Cohesin locates specific DNA sequences is currently unknown. To understand the role that SA1 or SA2 has in Cohesin/DNA interactions, we used the single-molecule techniques atomic force microscopy (AFM) and fluorescence imaging of quantum dot labeled proteins on DNA tightropes. Preliminary data indicates that SA1 carries out 1-D diffusion on DNA, binds with high affinity to telomeric sequences, and pauses on telomeric and promoter regions. In contrast, SA2 exhibits static and dynamic populations without pausing for telomere, centromere, and promoter DNA sequences. We propose that 1-D sliding and sequence dependent pausing by SA1 provides binding specificity and stability during the cohesion process at telomeres, while.SA2 alone, lacking the AT hook domain, uses different DNA binding mechanisms.}, number={2}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Countryman, Preston J. and Lin, Jiangguo and Kaur, Parminder and Brennan, Edward and Chen, Haijiang and You, Changjiang and Piehler, Jacob and Tao, Yizhi Jane and Wang, Hong}, year={2015}, month={Jan}, pages={397a} }
 @article{roushan_azad_lim_wang_riehn_2015, title={Interference of ATP with the fluorescent probes YOYO-1 and YOYO-3 modifies the mechanical properties of intercalator-stained DNA confined in nanochannels}, volume={182}, ISSN={0026-3672 1436-5073}, url={http://dx.doi.org/10.1007/S00604-015-1495-7}, DOI={10.1007/S00604-015-1495-7}, abstractNote={Intercalating fluorescent probes are widely used to visualize DNA in studies on DNA-protein interactions. Some require the presence of adenosine triphosphate (ATP). We have investigated the mechanical properties of DNA stained with the fluorescent intercalating dyes YOYO-1 and YOYO-3 as a function of ATP concentrations (up to 2 mM) by stretching single molecules in nanofluidic channels with a channel cross-section as small as roughly 100×100 nm2. The presence of ATP reduces the length of the DNA by up to 11 %. On the other hand, negligible effects are found if DNA is visualized with the minor groove-binding probe 4',6-diamidino-2-phenylindole. The apparent drop in extension under nanoconfinement is attributed to an interaction of the dye and ATP, and the resulting expulsion of YOYO-1 from the double helix.}, number={7-8}, journal={Microchimica Acta}, publisher={Springer Science and Business Media LLC}, author={Roushan, Maedeh and Azad, Zubair and Lim, Shuang Fang and Wang, Hong and Riehn, Robert}, year={2015}, month={Apr}, pages={1561–1565} }
 @article{wang_lin_kaur_countryman_opresko_smith_tao_2015, title={Revealing Structure and Dynamics of Telomere Maintenance Proteins on DNA: One Molecule at a Time}, volume={108}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2014.11.059}, DOI={10.1016/J.BPJ.2014.11.059}, abstractNote={Telomeres play important roles in maintaining the stability of linear chromosomes. A specialized protein complex, called shelterin or telosome, binds to and protects telomeres at chromosome ends. Telomere maintenance involves dynamic actions of multiple proteins interacting with long repetitive sequences and complex dynamic DNA structures, such as T-loops. Furthermore, it was shown recently that in contrast to cohesion along chromosome arms, sister telomere association is a specialized process requiring a tighter association provided by the cohesin subunit SA1 in conjunction with specific proteins from the shelterin complex. To better understand the telomere maintenance pathways, we established complementary single-molecule imaging platforms: a newly developed Dual-Resonance-frequency-Enhanced Electrostatic force Microscopy (DREEM) technique capable of revealing DNA paths in protein-DNA complexes, fluorescence imaging of quantum dot-labeled proteins for tracking dynamics of proteins on DNA tightropes, and a nanochannel based imaging platform for studying protein-mediated DNA-DNA pairing/looping in real time. I will highlight our recent results on: 1) Revealing DNA paths inside TRF2 complexes during DNA compaction through DREEM. 2) Cohesin SA1 and shelterin protein TRF1 mediated sister telomere cohesion. 3) Dynamics of SA1 and TRF1 mediated DNA-DNA pairing inside nanochannels.}, number={2}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Wang, Hong and Lin, Jiangguo and Kaur, Parminder and Countryman, Preston and Opresko, Patricia and Smith, Susan and Tao, Jane}, year={2015}, month={Jan}, pages={7a} }
 @article{lin_chen_kaur_miao_countryman_you_piehler_tao_smith_wang_2015, title={Single-Molecule Imaging Reveals Dynamics of SA1-TRF1 Interactions on Telomeric DNA}, volume={108}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2014.11.1138}, DOI={10.1016/J.BPJ.2014.11.1138}, abstractNote={The cohesin complex plays a crucial role in accurate chromosome segregation, organization of interphase chromatin, DNA replication, and post replicative DNA repair in part by promoting DNA-DNA pairing. The core cohesin subunits consist of a tripartite ring and the fourth core subunit Scc3/SA. In somatic vertebrate cells, SA can be either SA1 or SA2. Recent work indicates that while SA2 is important for cohesion at the centromere, SA1 plays a specific role in sister telomere association. In addition, SA1 directly interacts with shelterin subunits TRF1 and TIN2. While these results demonstrate a unique sister telomere cohesion process depending on the SA1-TRF1 complex, the underlying mechanism is still poorly understood. We applied Atomic Force Microscopy (AFM) and Total Internal Reflection Fluorescence Microscopy (TIRFM) to study the interactions between SA1 or SA1/TRF1 complex and various DNA substrates with or without telomeric sequences. DNA tightrope assays were performed and proteins were visualized by conjugating quantum dots. The data demonstrated that 1) SA1 carried out 1-dimensional diffusion on DNA substrate for searching telomeric DNA sequence; 2) SA1 paused at telomeric DNA sequence, while SA2 did not. Interestingly, the AFM data showed that SA1 further stabilized TRF1 mediated DNA-DNA pairing. These data shed more lights on the process of sister telomere association and segregation.}, number={2}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Lin, Jiangguo and Chen, Haijiang and Kaur, Parminder and Miao, Wang and Countryman, Preston and You, Changjiang and Piehler, Jacob and Tao, Yizhi J. and Smith, Susan and Wang, Hong}, year={2015}, month={Jan}, pages={206a} }
 @article{parikh_fouquerel_murphy_wang_opresko_2015, title={Telomeres are partly shielded from ultraviolet-induced damage and proficient for nucleotide excision repair of photoproducts}, volume={6}, ISSN={["2041-1723"]}, DOI={10.1038/ncomms9214}, abstractNote={Abstract Ultraviolet light induces cyclobutane pyrimidine dimers (CPD) and pyrimidine(6–4)pyrimidone photoproducts, which interfere with DNA replication and transcription. Nucleotide excision repair (NER) removes these photoproducts, but whether NER functions at telomeres is unresolved. Here we use immunospot blotting to examine the efficiency of photoproduct formation and removal at telomeres purified from UVC irradiated cells at various recovery times. Telomeres exhibit approximately twofold fewer photoproducts compared with the bulk genome in cells, and telomere-binding protein TRF1 significantly reduces photoproduct formation in telomeric fragments in vitro . CPD removal from telomeres occurs 1.5-fold faster than the bulk genome, and is completed by 48 h. 6–4PP removal is rapidly completed by 6 h in both telomeres and the overall genome. A requirement for XPA protein indicates the mechanism of telomeric photoproduct removal is NER. These data provide new evidence that telomeres are partially protected from ultraviolet irradiation and that NER preserves telomere integrity.}, journal={NATURE COMMUNICATIONS}, publisher={Nature Publishing Group}, author={Parikh, Dhvani and Fouquerel, Elise and Murphy, Connor T. and Wang, Hong and Opresko, Patricia L.}, year={2015}, month={Sep} }
 @article{roushan_kaur_karpusenko_countryman_ortiz_fang lim_wang_riehn_2014, title={Probing transient protein-mediated DNA linkages using nanoconfinement}, volume={8}, ISSN={1932-1058}, url={http://dx.doi.org/10.1063/1.4882775}, DOI={10.1063/1.4882775}, abstractNote={We present an analytic technique for probing protein-catalyzed transient DNA loops that is based on nanofluidic channels. In these nanochannels, DNA is forced in a linear configuration that makes loops appear as folds whose size can easily be quantified. Using this technique, we study the interaction between T4 DNA ligase and DNA. We find that T4 DNA ligase binding changes the physical characteristics of the DNA polymer, in particular persistence length and effective width. We find that the rate of DNA fold unrolling is significantly reduced when T4 DNA ligase and ATP are applied to bare DNA. Together with evidence of T4 DNA ligase bridging two different segments of DNA based on AFM imaging, we thus conclude that ligase can transiently stabilize folded DNA configurations by coordinating genetically distant DNA stretches.}, number={3}, journal={Biomicrofluidics}, publisher={AIP Publishing}, author={Roushan, Maedeh and Kaur, Parminder and Karpusenko, Alena and Countryman, Preston J. and Ortiz, Carlos P. and Fang Lim, Shuang and Wang, Hong and Riehn, Robert}, year={2014}, month={May}, pages={034113} }
 @article{ghodke_wang_hsieh_woldemeskel_watkins_rapic-otrin_van houten_2014, title={Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates}, volume={111}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.1323856111}, abstractNote={Significance UV damage in genomic DNA is identified by the human UV-damaged DNA-binding protein (UV-DDB). Recognition of DNA damage by UV-DDB serves to initiate global genomic nucleotide excision repair (NER) in humans. Recent work has revealed that UV-DDB dimerizes at sites of damage. This study demonstrates that prior to stable damage recognition, UV-DDB interrogates DNA for damage via a 3D diffusion mechanism coupled to the formation of multiple transient intermediates. Stable binding at sites of damage is achieved by dimerization of UV-DDB. This study also analyzed a disease-causing mutant of UV-DDB, which was found to slide on DNA, while retaining the ability to dimerize on DNA. These results enhance our understanding of damage recognition in NER in humans.}, number={18}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, publisher={National Acad Sciences}, author={Ghodke, Harshad and Wang, Hong and Hsieh, Ching L. and Woldemeskel, Selamawit and Watkins, Simon C. and Rapic-Otrin, Vesna and Van Houten, Bennett}, year={2014}, month={May}, pages={E1862–E1871} }
 @inproceedings{lin_kaur_chen_countryman_roushan_flaherty_brennan_you_piehler_riehn_et al._2014, title={Single-molecule imaging reveals DNA-binding properties of cohesin proteins SA1 and SA2.}, volume={55}, booktitle={Environmental and Molecular Mutagenesis}, author={Lin, J. and Kaur, P. and Chen, H. and Countryman, P. and Roushan, M. and Flaherty, D. and Brennan, E. and You, C. and Piehler, J. and Riehn, R. and et al.}, year={2014}, pages={S29–29} }
 @article{lin_countryman_buncher_kaur_longjiang_zhang_gibson_you_watkins_piehler_et al._2014, title={TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres}, volume={42}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkt1132}, abstractNote={Human telomeres are maintained by the shelterin protein complex in which TRF1 and TRF2 bind directly to duplex telomeric DNA. How these proteins find telomeric sequences among a genome of billions of base pairs and how they find protein partners to form the shelterin complex remains uncertain. Using single-molecule fluorescence imaging of quantum dot-labeled TRF1 and TRF2, we study how these proteins locate TTAGGG repeats on DNA tightropes. By virtue of its basic domain TRF2 performs an extensive 1D search on nontelomeric DNA, whereas TRF1's 1D search is limited. Unlike the stable and static associations observed for other proteins at specific binding sites, TRF proteins possess reduced binding stability marked by transient binding (∼ 9-17 s) and slow 1D diffusion on specific telomeric regions. These slow diffusion constants yield activation energy barriers to sliding ∼ 2.8-3.6 κ(B)T greater than those for nontelomeric DNA. We propose that the TRF proteins use 1D sliding to find protein partners and assemble the shelterin complex, which in turn stabilizes the interaction with specific telomeric DNA. This 'tag-team proofreading' represents a more general mechanism to ensure a specific set of proteins interact with each other on long repetitive specific DNA sequences without requiring external energy sources.}, number={4}, journal={NUCLEIC ACIDS RESEARCH}, author={Lin, Jiangguo and Countryman, Preston and Buncher, Noah and Kaur, Parminder and Longjiang, E. and Zhang, Yiyun and Gibson, Greg and You, Changjiang and Watkins, Simon C. and Piehler, Jacob and et al.}, year={2014}, month={Feb}, pages={2493–2504} }
 @article{lin_countryman_buncher_kaur_longjiang_zhang_gibson_you_watkins_piehler_et al._2014, title={TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres}, volume={42}, number={4}, journal={Nucleic acids research}, publisher={Oxford Univ Press}, author={Lin, Jiangguo and Countryman, Preston and Buncher, Noah and Kaur, Parminder and Longjiang, E and Zhang, Yiyun and Gibson, Greg and You, Changjiang and Watkins, Simon C and Piehler, Jacob and et al.}, year={2014}, pages={2493–2504} }
 @article{lin_kaur_countryman_opresko_wang_2014, title={Unraveling secrets of telomeres: One molecule at a time}, volume={20}, ISSN={["1568-7856"]}, DOI={10.1016/j.dnarep.2014.01.012}, abstractNote={Telomeres play important roles in maintaining the stability of linear chromosomes. Telomere maintenance involves dynamic actions of multiple proteins interacting with long repetitive sequences and complex dynamic DNA structures, such as G-quadruplexes, T-loops and t-circles. Given the heterogeneity and complexity of telomeres, single-molecule approaches are essential to fully understand the structure-function relationships that govern telomere maintenance. In this review, we present a brief overview of the principles of single-molecule imaging and manipulation techniques. We then highlight results obtained from applying these single-molecule techniques for studying structure, dynamics and functions of G-quadruplexes, telomerase, and shelterin proteins.}, journal={DNA REPAIR}, author={Lin, Jiangguo and Kaur, Parminder and Countryman, Preston and Opresko, Patricia L. and Wang, Hong}, year={2014}, month={Aug}, pages={142–153} }
 @article{lormand_buncher_murphy_kaur_lee_burgers_wang_kunkel_opresko_2013, title={DNA polymerase delta stalls on telomeric lagging strand templates independently from G-quadruplex formation}, volume={41}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkt813}, abstractNote={Previous evidence indicates that telomeres resemble common fragile sites and present a challenge for DNA replication. The precise impediments to replication fork progression at telomeric TTAGGG repeats are unknown, but are proposed to include G-quadruplexes (G4) on the G-rich strand. Here we examined DNA synthesis and progression by the replicative DNA polymerase δ/proliferating cell nuclear antigen/replication factor C complex on telomeric templates that mimic the leading C-rich and lagging G-rich strands. Increased polymerase stalling occurred on the G-rich template, compared with the C-rich and nontelomeric templates. Suppression of G4 formation by substituting Li(+) for K(+) as the cation, or by using templates with 7-deaza-G residues, did not alleviate Pol δ pause sites within the G residues. Furthermore, we provide evidence that G4 folding is less stable on single-stranded circular TTAGGG templates where ends are constrained, compared with linear oligonucleotides. Artificially stabilizing G4 structures on the circular templates with the G4 ligand BRACO-19 inhibited Pol δ progression into the G-rich repeats. Similar results were obtained for yeast and human Pol δ complexes. Our data indicate that G4 formation is not required for polymerase stalling on telomeric lagging strands and suggest that an alternative mechanism, in addition to stable G4s, contributes to replication stalling at telomeres.}, number={22}, journal={NUCLEIC ACIDS RESEARCH}, publisher={Oxford Univ Press}, author={Lormand, Justin D. and Buncher, Noah and Murphy, Connor T. and Kaur, Parminder and Lee, Marietta Y. and Burgers, Peter and Wang, Hong and Kunkel, Thomas A. and Opresko, Patricia L.}, year={2013}, month={Dec}, pages={10323–10333} }
 @misc{tessmer_kaur_lin_wang_2013, title={Investigating bioconjugation by atomic force microscopy}, volume={11}, journal={Journal of Anobiotechnology}, author={Tessmer, I. and Kaur, P. and Lin, J. G. and Wang, H.}, year={2013} }
 @article{tessmer_kaur_lin_wang_2013, title={Investigating bioconjugation by atomic force microscopy}, volume={11}, journal={J. Nanobiotechnol}, author={Tessmer, Ingrid and Kaur, Parminder and Lin, Jiangguo and Wang, Hong}, year={2013}, pages={25} }
 @article{hughes_wang_ghodke_simons_towheed_peng_van houten_kad_2013, title={Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes}, journal={Nucleic acids research}, publisher={Oxford Univ Press}, author={Hughes, Craig D and Wang, Hong and Ghodke, Harshad and Simons, Michelle and Towheed, Atif and Peng, Ye and Van Houten, Bennett and Kad, Neil M}, year={2013}, pages={gkt177} }
 @article{ghodke_wang_hsieh_gibson_watkins_rapic-otrin_levine_van houten_2013, title={WT UV-DDB Performs a 3D Search on DNA whereas the XP-E Mutant (K244E DDB2) Mutant Slides}, volume={104}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/j.bpj.2012.11.462}, DOI={10.1016/j.bpj.2012.11.462}, abstractNote={The DNA damage binding protein complex (UV-DDB) recognizes ultraviolet light (UV) induced lesions such as 6-4 photoproducts (6-4PPs) and cyclobutane pyrimidine dimers (CPDs) in DNA and initiates human nucleotide excision repair in chromatin. Crystallographic studies have revealed that UV-DDB binds to damaged DNA as a heterodimer of DDB1 and DDB2 on short DNA substrates; however, its oligomeric state on longer, physiologically relevant substrates and on nucleosomes remains undetermined. Additionally, the question of how UV-DDB searches a sea of undamaged chromatin for UV-induced lesions remains unresolved. We assayed purified UV-DDB for binding to a 517 bp UV-irradiated, PCR fragment using atomic force microscopy (AFM). Volume analysis revealed that UV-DDB binds primarily as a dimer of heterodimers to UV damaged DNA, with 19% of these UV-DDB dimers binding simultaneously to two DNA molecules In order to study damage recognition in real time, we used a His-tag on UV-DDB (either DDB1 or DDB2) to conjugate quantum dots(QDs). QD-UV-DDB retained DNA damage binding activity, as assayed by electrophoretic mobility shift assays. To identify the search mode, we have employed an oblique angle fluorescence microscopy setup to track single molecules of QD tagged UV-DDB on UV-damaged DNA tightropes. We have identified that WT UV-DDB employs a 3D search to identify DNA damage, with a long residence time when bound to sites of damage. Consistent with several salt-bridges observed in the co-crystal structure, we have found that the mobility of UV-DDB on DNA is salt dependent. Further, we have assayed the disease causing K244E mutant of DDB2 and observed that the mutant retains DNA binding activity, but slides on DNA compared to the WT. Our results reveal the stoichiometry and search mechanism of UV-DDB in damage surveillance.}, number={2}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Ghodke, Harshad and Wang, Hong and Hsieh, Ching L. and Gibson, Gregory and Watkins, Simon and Rapic-Otrin, Vesna and Levine, Arthur S. and Van Houten, Bennett}, year={2013}, month={Jan}, pages={77a} }
 @article{hughes_towheed_simons_wang_van houten_kad_2012, title={A New Role for UvrB in NER? Single Molecule Imaging of the NER Complex UvrBC-DNA}, volume={102}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/j.bpj.2011.11.2659}, DOI={10.1016/j.bpj.2011.11.2659}, abstractNote={Direct observation of a multi-protein DNA repair pathway is now possible using single molecule methods. Previously we have shown that UvrA and UvrB belonging to the bacterial nucleotide excision repair pathway collaborate to locate damage. Here we report on the next phase of repair, dual incision by UvrC. However as UvrC is in such short supply in the cell, locating a pre-incision complex at a site of damage becomes the key rate limiting step of NER. By labelling UvrB and UvrC with different coloured quantum dots their interactions with DNA tightropes can be studied at the single molecule level using oblique angle fluorescence microscopy. We found UvrC interacts with DNA and performs a 1D diffusional search (7.4x10−2 μm2s−1). Surprisingly, we also found that UvrC could load UvrB onto DNA generating a previously unseen UvrBC-DNA complex. This UvrBC complex is highly mobile relative to UvrC alone (15% vs. 57% mobility) and engages in unbiased 1D diffusion with a coefficient of 6.6x10−3 μm2s−1. Ionic strength profoundly affects the motion of the proteins; at elevated salt more UvrC molecules slide, however UvrBC mobility remains constant. Furthermore, the inclusion of ATP decreases UvrBC's dwell time on DNA from 175s to 83s. Based on these and further results from the study of various mutants we deduce that UvrB switches UvrC's search mechanism from 3D distributive to 1D sliding. We propose a new chaperoning role for UvrB in NER, where it protects the genome from unwanted UvrC nuclease activity but facilitates UvrC's location of pre-incision UvrB complexes.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Hughes, Craig D. and Towheed, Mohammed A. and Simons, Michelle and Wang, Hong and Van Houten, Bennett and Kad, Neil M.}, year={2012}, month={Jan}, pages={485a} }
 @article{yeh_levine_du_chinte_ghodke_wang_shi_hsieh_conway_van houten_et al._2012, title={Damaged DNA induced UV-damaged DNA-binding protein (UV-DDB) dimerization and its roles in chromatinized DNA repair}, volume={109}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.1110067109}, DOI={10.1073/pnas.1110067109}, abstractNote={UV light-induced photoproducts are recognized and removed by the nucleotide-excision repair (NER) pathway. In humans, the UV-damaged DNA-binding protein (UV-DDB) is part of a ubiquitin E3 ligase complex (DDB1-CUL4A DDB2 ) that initiates NER by recognizing damaged chromatin with concomitant ubiquitination of core histones at the lesion. We report the X-ray crystal structure of the human UV-DDB in a complex with damaged DNA and show that the N-terminal domain of DDB2 makes critical contacts with two molecules of DNA, driving N-terminal-domain folding and promoting UV-DDB dimerization. The functional significance of the dimeric UV-DDB [(DDB1-DDB2) 2 ], in a complex with damaged DNA, is validated by electron microscopy, atomic force microscopy, solution biophysical, and functional analyses. We propose that the binding of UV-damaged DNA results in conformational changes in the N-terminal domain of DDB2, inducing helical folding in the context of the bound DNA and inducing dimerization as a function of nucleotide binding. The temporal and spatial interplay between domain ordering and dimerization provides an elegant molecular rationale for the unprecedented binding affinities and selectivities exhibited by UV-DDB for UV-damaged DNA. Modeling the DDB1-CUL4A DDB2 complex according to the dimeric UV-DDB-AP24 architecture results in a mechanistically consistent alignment of the E3 ligase bound to a nucleosome harboring damaged DNA. Our findings provide unique structural and conformational insights into the molecular architecture of the DDB1-CUL4A DDB2 E3 ligase, with significant implications for the regulation and overall organization of the proteins responsible for initiation of NER in the context of chromatin and for the consequent maintenance of genomic integrity.}, number={41}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Yeh, J. I. and Levine, A. S. and Du, S. and Chinte, U. and Ghodke, H. and Wang, H. and Shi, H. and Hsieh, C. L. and Conway, J. F. and Van Houten, B. and et al.}, year={2012}, month={Jul}, pages={E2737–E2746} }
 @article{damerla_knickelbein_strutt_liu_wang_opresko_2012, title={Werner syndrome protein suppresses the formation of large deletions during the replication of human telomeric sequences}, volume={11}, number={16}, journal={Cell cycle}, publisher={Taylor & Francis}, author={Damerla, Rama Rao and Knickelbein, Kelly E and Strutt, Steven and Liu, Fu-Jun and Wang, Hong and Opresko, Patricia L}, year={2012}, pages={3036–3044} }
 @article{fronczek_quammen_wang_kisker_superfine_taylor_erie_tessmer_2011, title={High accuracy FIONA–AFM hybrid imaging}, volume={111}, ISSN={0304-3991}, url={http://dx.doi.org/10.1016/j.ultramic.2011.01.020}, DOI={10.1016/j.ultramic.2011.01.020}, abstractNote={Multi-protein complexes are ubiquitous and play essential roles in many biological mechanisms. Single molecule imaging techniques such as electron microscopy (EM) and atomic force microscopy (AFM) are powerful methods for characterizing the structural properties of multi-protein and multi-protein–DNA complexes. However, a significant limitation to these techniques is the ability to distinguish different proteins from one another. Here, we combine high resolution fluorescence microscopy and AFM (FIONA–AFM) to allow the identification of different proteins in such complexes. Using quantum dots as fiducial markers in addition to fluorescently labeled proteins, we are able to align fluorescence and AFM information to ≥8 nm accuracy. This accuracy is sufficient to identify individual fluorescently labeled proteins in most multi-protein complexes. We investigate the limitations of localization precision and accuracy in fluorescence and AFM images separately and their effects on the overall registration accuracy of FIONA–AFM hybrid images. This combination of the two orthogonal techniques (FIONA and AFM) opens a wide spectrum of possible applications to the study of protein interactions, because AFM can yield high resolution (5–10 nm) information about the conformational properties of multi-protein complexes and the fluorescence can indicate spatial relationships of the proteins in the complexes.}, number={5}, journal={Ultramicroscopy}, publisher={Elsevier BV}, author={Fronczek, D.N. and Quammen, C. and Wang, H. and Kisker, C. and Superfine, R. and Taylor, R. and Erie, D.A. and Tessmer, I.}, year={2011}, month={Apr}, pages={350–355} }
 @article{keller_wang_towheed_van houten_kad_2011, title={Investigating Nucleotide Excision DNA Repair by Single-Molecule Imaging of Quantum Dot Labeled Proteins Reveals Unique Scanning Mechanisms}, volume={100}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/j.bpj.2010.12.1531}, DOI={10.1016/j.bpj.2010.12.1531}, abstractNote={How DNA repair proteins locate and repair lesions amongst a vast excess of undamaged DNA is a key question for cell survival across all kingdoms of life. Previously, using a newly developed single molecule approach we studied how the emergent properties of the first two enzymes (UvrA & UvrB) of the prokaryotic Nucleotide Excision Repair system made this lesion search possible. In this study we have introduced the next enzyme in the pathway, UvrC, which nicks the DNA backbone 5’ and 3’ to the pre-incision complex formed after UvrAB finds the lesion. This provides a substrate for DNA helicase II which subsequently removes the damage-containing oligonucleotide. We have cloned and expressed UvrC with an avi-tag that covalently links to biotin thus providing a conjugation moiety for quantum dot attachment. Electrophoretic mobility shift assays indicate that tagged UvrC is still able to bind DNA and AFM data suggest that UvrC is a monomer. Initial fluorescence results using our single molecule ‘tightrope’ approach indicate that when a color mixed population of UvrC is investigated there is little colocalization, confirming a monomer is capable of binding to DNA. The interaction of UvrC with DNA shows an interesting pattern of a short period of 1D diffusion before halting on the DNA. However, in the presence of UvrB we observe that the diffusive motion persists for the duration of attachment. Not only does this suggest that UvrB alters the search mechanism of UvrC, as it does for UvrA, but also that UvrC is capable of bringing UvrB to the DNA. Physiologically, UvrC may take advantage of the cellular excess of UvrB to enhance its diffusive search for other UvrB molecules trapped in pre-incision complexes.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Keller, Robert and Wang, Hong and Towheed, Mohammed A. and Van Houten, Bennett and Kad, Neil M.}, year={2011}, month={Feb}, pages={240a} }
 @inbook{peng_wang_santana-santos_kisker_van houten_2011, title={Nucleotide excision repair from bacteria to humans: Structure--function studies}, booktitle={Chemical Carcinogenesis}, publisher={Springer}, author={Peng, Ye and Wang, Hong and Santana-Santos, Lucas and Kisker, Caroline and Van Houten, Bennett}, year={2011}, pages={267–296} }
 @article{wang_nora_ghodke_opresko_2011, title={Single Molecule Studies of Physiologically Relevant Telomeric Tails Reveal POT1 Mechanism for Promoting G-quadruplex Unfolding}, volume={286}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/jbc.M110.205641}, DOI={10.1074/jbc.M110.205641}, abstractNote={Human telomeres are composed of duplex TTAGGG repeats and a 3′ single-stranded DNA tail. The telomeric DNA is protected and regulated by the shelterin proteins, including the protection of telomeres 1 (POT1) protein that binds telomeric single-stranded DNA. The single-stranded tail can fold into G-quadruplex (G4) DNA. Both POT1 and G4 DNA play important roles in regulating telomere length homeostasis. To date, most studies have focused on individual quadruplexes formed by four TTAGGG repeats. Telomeric tails in human cells have on average six times as many repeats, and no structural studies have examined POT1 binding in competition with G4 DNA folding. Using single molecule atomic force microscopy imaging, we observed that the majority of the telomeric tails of 16 repeats formed two quadruplexes even though four were possible. The result that physiological telomeric tails rarely form the maximum potential number of G4 units provides a structural basis for the coexistence of G4 and POT1 on the same DNA molecule, which is observed directly in the captured atomic force microscopy images. We further observed that POT1 is significantly more effective in disrupting quadruplex DNA on long telomeric tails than an antisense oligonucleotide, indicating a novel POT1 activity beyond simply preventing quadruplex folding. Human telomeres are composed of duplex TTAGGG repeats and a 3′ single-stranded DNA tail. The telomeric DNA is protected and regulated by the shelterin proteins, including the protection of telomeres 1 (POT1) protein that binds telomeric single-stranded DNA. The single-stranded tail can fold into G-quadruplex (G4) DNA. Both POT1 and G4 DNA play important roles in regulating telomere length homeostasis. To date, most studies have focused on individual quadruplexes formed by four TTAGGG repeats. Telomeric tails in human cells have on average six times as many repeats, and no structural studies have examined POT1 binding in competition with G4 DNA folding. Using single molecule atomic force microscopy imaging, we observed that the majority of the telomeric tails of 16 repeats formed two quadruplexes even though four were possible. The result that physiological telomeric tails rarely form the maximum potential number of G4 units provides a structural basis for the coexistence of G4 and POT1 on the same DNA molecule, which is observed directly in the captured atomic force microscopy images. We further observed that POT1 is significantly more effective in disrupting quadruplex DNA on long telomeric tails than an antisense oligonucleotide, indicating a novel POT1 activity beyond simply preventing quadruplex folding.}, number={9}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Wang, Hong and Nora, Gerald J. and Ghodke, Harshad and Opresko, Patricia L.}, year={2011}, month={Mar}, pages={7479–7489} }
 @article{kad_wang_kennedy_warshaw_van houten_2010, title={Collaborative Dynamic DNA Scanning by Nucleotide Excision Repair Proteins Investigated by Single- Molecule Imaging of Quantum-Dot-Labeled Proteins}, volume={37}, ISSN={1097-2765}, url={http://dx.doi.org/10.1016/j.molcel.2010.02.003}, DOI={10.1016/j.molcel.2010.02.003}, abstractNote={How DNA repair proteins sort through a genome for damage is one of the fundamental unanswered questions in this field. To address this problem, we uniquely labeled bacterial UvrA and UvrB with differently colored quantum dots and visualized how they interacted with DNA individually or together using oblique-angle fluorescence microscopy. UvrA was observed to utilize a three-dimensional search mechanism, binding transiently to the DNA for short periods (7 s). UvrA also was observed jumping from one DNA molecule to another over ∼1 μm distances. Two UvrBs can bind to a UvrA dimer and collapse the search dimensionality of UvrA from three to one dimension by inducing a substantial number of UvrAB complexes to slide along the DNA. Three types of sliding motion were characterized: random diffusion, paused motion, and directed motion. This UvrB-induced change in mode of searching permits more rapid and efficient scanning of the genome for damage.}, number={5}, journal={Molecular Cell}, publisher={Elsevier BV}, author={Kad, Neil M. and Wang, Hong and Kennedy, Guy G. and Warshaw, David M. and Van Houten, Bennett}, year={2010}, month={Mar}, pages={702–713} }
 @article{sowd_wang_pretto_chazin_opresko_2009, title={Replication Protein A Stimulates the Werner Syndrome Protein Branch Migration Activity}, volume={284}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/jbc.M109.049031}, DOI={10.1074/jbc.M109.049031}, abstractNote={Loss of the RecQ DNA helicase WRN protein causes Werner syndrome, in which patients exhibit features of premature aging and increased cancer. WRN deficiency induces cellular defects in DNA replication, mitotic homologous recombination (HR), and telomere stability. In addition to DNA unwinding activity, WRN also possesses exonuclease, strand annealing, and branch migration activities. The single strand binding proteins replication protein A (RPA) and telomere-specific POT1 specifically stimulate WRN DNA unwinding activity. To determine whether RPA and POT1 also modulate WRN branch migration activity, we examined biologically relevant mobile D-loops that mimic structures in HR strand invasion and at telomere ends. Both RPA and POT1 block WRN exonuclease digestion of the invading strand by loading on the strand. However, only RPA robustly stimulates WRN branch migration activity and increases the percentage of D-loops that are disrupted. Our results are consistent with cellular data that support RPA enhancement of branch migration during HR repair and, conversely, POT1 limitation of inappropriate recombination and branch migration at telomeric ends. This is, to our knowledge, the first evidence that RPA can stimulate branch migration activity.}, number={50}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Sowd, Gregory and Wang, Hong and Pretto, Dalyir and Chazin, Walter J. and Opresko, Patricia L.}, year={2009}, month={Oct}, pages={34682–34691} }
 @article{opresko_sowd_wang_2009, title={The Werner syndrome helicase/exonuclease processes mobile D-loops through branch migration and degradation}, volume={4}, number={3}, journal={PLoS One}, publisher={Public Library of Science}, author={Opresko, Patricia L and Sowd, Gregory and Wang, Hong}, year={2009}, pages={e4825} }
 @article{christensen_wang_van houten_vasquez_2008, title={Efficient processing of TFO-directed psoralen DNA interstrand crosslinks by the UvrABC nuclease}, volume={36}, number={22}, journal={Nucleic acids research}, publisher={Oxford Univ Press}, author={Christensen, Laura A and Wang, Hong and Van Houten, Bennett and Vasquez, Karen M}, year={2008}, pages={7136–7145} }
 @article{wang_tessmer_croteau_erie_van houten_2008, title={Functional Characterization and Atomic Force Microscopy of a DNA Repair Protein Conjugated to a Quantum Dot}, volume={8}, ISSN={1530-6984 1530-6992}, url={http://dx.doi.org/10.1021/nl080316l}, DOI={10.1021/nl080316l}, abstractNote={Quantum dots (QDs) possess highly desirable optical properties that make them ideal fluorescent labels for studying the dynamic behavior of proteins. However, a lack of characterization methods for reliably determining protein−quantum dot conjugate stoichiometry and functionality has impeded their widespread use in single-molecule studies. We used atomic force microscopic (AFM) imaging to demonstrate the 1:1 formation of UvrB−QD conjugates based on an antibody-sandwich method. We show that an agarose gel-based electrophoresis mobility shift assay and AFM can be used to evaluate the DNA binding function of UvrB−QD conjugates. Importantly, we demonstrate that quantum dots can serve as a molecular marker to unambiguously identify the presence of a labeled protein in AFM images.}, number={6}, journal={Nano Letters}, publisher={American Chemical Society (ACS)}, author={Wang, Hong and Tessmer, Ingrid and Croteau, Deborah L. and Erie, Dorothy A. and Van Houten, Bennett}, year={2008}, month={Jun}, pages={1631–1637} }
 @inbook{wang_yang_erie_2007, title={Characterization of protein--protein interactions using atomic force microscopy}, booktitle={Protein Interactions}, publisher={Springer}, author={Wang, Hong and Yang, Yong and Erie, Dorothy A}, year={2007}, pages={39–77} }
 @article{truglio_karakas_rhau_wang_dellavecchia_van houten_kisker_2006, title={Structural basis for DNA recognition and processing by UvrB}, volume={13}, ISSN={1545-9993 1545-9985}, url={http://dx.doi.org/10.1038/nsmb1072}, DOI={10.1038/nsmb1072}, abstractNote={DNA-damage recognition in the nucleotide excision repair (NER) cascade is a complex process, operating on a wide variety of damages. UvrB is the central component in prokaryotic NER, directly involved in DNA-damage recognition and guiding the DNA through repair synthesis. We report the first structure of a UvrB-double-stranded DNA complex, providing insights into the mechanism by which UvrB binds DNA, leading to formation of the preincision complex. One DNA strand, containing a 3' overhang, threads behind a beta-hairpin motif of UvrB, indicating that this motif inserts between the strands of the double helix, thereby locking down either the damaged or undamaged strand. The nucleotide directly behind the beta-hairpin is flipped out and inserted into a small, highly conserved pocket in UvrB.}, number={4}, journal={Nature Structural & Molecular Biology}, publisher={Springer Science and Business Media LLC}, author={Truglio, James J and Karakas, Erkan and Rhau, Benjamin and Wang, Hong and DellaVecchia, Matthew J and Van Houten, Bennett and Kisker, Caroline}, year={2006}, month={Mar}, pages={360–364} }
 @article{croteau_dellavecchia_wang_bienstock_melton_van houten_2006, title={The C-terminal Zinc Finger of UvrA Does Not Bind DNA Directly but Regulates Damage-specific DNA Binding}, volume={281}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/jbc.M603093200}, DOI={10.1074/jbc.M603093200}, abstractNote={In prokaryotic nucleotide excision repair, UvrA recognizes DNA perturbations and recruits UvrB for the recognition and processing steps in the reaction. One of the most remarkable aspects of UvrA is that it can recognize a wide range of DNA lesions that differ in chemistry and structure. However, how UvrA interacts with DNA is unknown. To examine the role that the UvrA C-terminal zinc finger domain plays in DNA binding, an eleven amino acid deletion was constructed (ZnG UvrA). Biochemical characterization of the ZnG UvrA protein was carried out using UvrABC DNA incision, DNA binding and ATPase assays. Although ZnG UvrA was able to bind dsDNA slightly better than wild-type UvrA, the ZnG UvrA mutant only supported 50-75% of wild type incision. Surprisingly, the ZnG UvrA mutant, while retaining its ability to bind dsDNA, did not support damage-specific binding. Furthermore, this mutant protein only provided 10% of wild-type Bca UvrA complementation for UV survival of an uvrA deletion strain. In addition, ZnG UvrA failed to stimulate the UvrB DNA damage-associated ATPase activity. Electrophoretic mobility shift analysis was used to monitor UvrB loading onto damaged DNA with wild-type UvrA or ZnG UvrA. The ZnG UvrA protein showed a 30-60% reduction in UvrB loading as compared with the amount of UvrB loaded by wild-type UvrA. These data demonstrate that the C-terminal zinc finger of UvrA is required for regulation of damage-specific DNA binding. In prokaryotic nucleotide excision repair, UvrA recognizes DNA perturbations and recruits UvrB for the recognition and processing steps in the reaction. One of the most remarkable aspects of UvrA is that it can recognize a wide range of DNA lesions that differ in chemistry and structure. However, how UvrA interacts with DNA is unknown. To examine the role that the UvrA C-terminal zinc finger domain plays in DNA binding, an eleven amino acid deletion was constructed (ZnG UvrA). Biochemical characterization of the ZnG UvrA protein was carried out using UvrABC DNA incision, DNA binding and ATPase assays. Although ZnG UvrA was able to bind dsDNA slightly better than wild-type UvrA, the ZnG UvrA mutant only supported 50-75% of wild type incision. Surprisingly, the ZnG UvrA mutant, while retaining its ability to bind dsDNA, did not support damage-specific binding. Furthermore, this mutant protein only provided 10% of wild-type Bca UvrA complementation for UV survival of an uvrA deletion strain. In addition, ZnG UvrA failed to stimulate the UvrB DNA damage-associated ATPase activity. Electrophoretic mobility shift analysis was used to monitor UvrB loading onto damaged DNA with wild-type UvrA or ZnG UvrA. The ZnG UvrA protein showed a 30-60% reduction in UvrB loading as compared with the amount of UvrB loaded by wild-type UvrA. These data demonstrate that the C-terminal zinc finger of UvrA is required for regulation of damage-specific DNA binding. Nucleotide excision repair (NER) 2The abbreviations used are: NER, nucleotide excision repair; ABC ATPase, ATP-binding cassette ATPase; CABC, UvrA C-terminal ABC ATPase domain; dsDNA, double-stranded DNA; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; SC DNA, supercoiled DNA; UV DNA, UV-irradiated DNA; IPTG, isopropyl-1-thio-β-d-galactopyranoside; DTT, dithiothreitol; Wt, wild type; Bca, Bacillus caldotenax; RMS, root mean-square; PDB, Protein Data Bank.2The abbreviations used are: NER, nucleotide excision repair; ABC ATPase, ATP-binding cassette ATPase; CABC, UvrA C-terminal ABC ATPase domain; dsDNA, double-stranded DNA; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; SC DNA, supercoiled DNA; UV DNA, UV-irradiated DNA; IPTG, isopropyl-1-thio-β-d-galactopyranoside; DTT, dithiothreitol; Wt, wild type; Bca, Bacillus caldotenax; RMS, root mean-square; PDB, Protein Data Bank. is the primary mechanism cells use to repair a diverse set of DNA lesions. In prokaryotes, nucleotide excision repair requires the collaborative action of UvrA, UvrB, and UvrC. The fundamental process of DNA damage recognition is central to the function of UvrA and the overall repair reaction. It is believed that UvrA initially recognizes the damage-induced distortion in the DNA, and then hands off the damaged DNA to UvrB, so that UvrB can make a more detailed assessment of the nature of the helical perturbation. Once on DNA, UvrB is responsible for preparing the DNA for incision by the two nuclease centers of UvrC (for recent reviews see Refs. 1Van Houten B. Croteau D.L. DellaVecchia M.J. Wang H. Kisker C. Mutat. Res. 2005; 577: 92-117Crossref PubMed Scopus (113) Google Scholar, 2Truglio J.J. Croteau D.L. Van Houten B. Kisker C. Chem. Rev. 2006; 106: 233-252Crossref PubMed Scopus (257) Google Scholar, 3Croteau D.L. Dellavecchia M.J. Skorvaga M. Van Houten B. Wolfram S. Kow Y.W. Doetsch P.W. DNA Damage Recognition. Taylor & Francis, Boca Raton, FL2005: 111-138Google Scholar). In a general way, NER proteins fulfill all the criteria of a kinetic proofreading mechanism for damage processing: 1) damage specificity is not absolute; 2) ATP is consumed to generate irreversible intermediates and to delay initial binding and processing from incision, and 3) dissociation of UvrA or UvrA/UvrB from DNA or lack of stimulating the UvrC nuclease domains help safeguard against inappropriate incision (4Reardon J.T. Sancar A. Cell Cycle. 2004; 3: 141-144Crossref PubMed Scopus (2) Google Scholar). However, the precise mechanism by which UvrA and UvrB effectively recognize DNA damage in a sea of non-damaged DNA is unknown and of critical importance.UvrA is a large protein, ∼105 kDa, possessing two ATP-binding cassette-type ATPases (ABC ATPase) and two zinc finger domains (5Doolittle R.F. Johnson M.S. Husain I. Van Houten B. Thomas D.C. Sancar A. Nature. 1986; 323: 451-453Crossref PubMed Scopus (111) Google Scholar, 6Navaratnam S. Myles G.M. Strange R.W. Sancar A. J. Biol. Chem. 1989; 264: 16067-16071Abstract Full Text PDF PubMed Google Scholar) and was recently reviewed in Ref. 3Croteau D.L. Dellavecchia M.J. Skorvaga M. Van Houten B. Wolfram S. Kow Y.W. Doetsch P.W. DNA Damage Recognition. Taylor & Francis, Boca Raton, FL2005: 111-138Google Scholar. The conserved motifs of the ABC ATPase are not contiguous, but are interrupted after the Walker A and before the signature sequence, LSGG, with an insertion sequence containing the conserved zinc fingers. The two ABC ATPases are connected through a flexible protease-sensitive linker region. Therefore, both the N- and C-terminal ABC ATPase domains are separable, and each has been shown to possess some DNA binding capacity (7Myles G.M. Sancar A. Biochemistry. 1991; 30: 3834-3840Crossref PubMed Scopus (27) Google Scholar). Because of the presence of the ATPase domains, DNA binding by UvrA is regulated by ATP binding and hydrolysis. Whereas nucleotide binding by UvrA is not absolutely required for DNA binding, ATP binding promotes the dimerization of UvrA (8Orren D.K. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5237-5241Crossref PubMed Scopus (167) Google Scholar), which in turn facilitates DNA binding (9Mazur S. Grossman L. Biochemistry. 1991; 30: 4432-4443Crossref PubMed Scopus (83) Google Scholar). In contrast, ATP hydrolysis is believed to drive UvrA dimer dissociation and consequently reduces DNA binding (10Reardon J.T. Nichols A.F. Keeney S. Smith C.A. Taylor J.S. Linn S. Sancar A. J. Biol. Chem. 1993; 268: 21301-21308Abstract Full Text PDF PubMed Google Scholar, 11Thiagalingam S. Grossman L. J. Biol. Chem. 1993; 268: 18382-18389Abstract Full Text PDF PubMed Google Scholar). Despite this general picture, the precise mechanistic details of how nucleotide binding and hydrolysis regulate the UvrA DNA binding are not known.Zinc fingers are a common structural element utilized by sequence-specific DNA-binding proteins to interact with DNA (12Berg J.M. Shi Y. Science. 1996; 271: 1081-1085Crossref PubMed Scopus (1659) Google Scholar). However, zinc fingers also mediate protein-protein, protein-RNA, and protein-ligand interactions (13Matthews J.M. Sunde M. IUBMB Life. 2002; 54: 351-355Crossref PubMed Scopus (240) Google Scholar). Currently, there are 64 families of zinc fingers listed in the PROSITE data base, yet neither of UvrA zinc fingers shares strong homology to any of these family members. Previously, investigators mutagenized the zinc-coordinating cysteines of Escherichia coli UvrA C-terminal zinc finger producing bacteria that were UV-sensitive and NER-defective (14Visse R. de Ruijter M. Ubbink M. Brandsma J.A. van de Putte P. Mutat. Res. 1993; 294: 263-274Crossref PubMed Scopus (22) Google Scholar, 15Wang J. Grossman L. J. Biol. Chem. 1993; 268: 5323-5331Abstract Full Text PDF PubMed Google Scholar, 16Wang J. Mueller K.L. Grossman L. J. Biol. Chem. 1994; 269: 10771-10775Abstract Full Text PDF PubMed Google Scholar).In this study, we re-investigated the role of the UvrA C-terminal zinc finger in the NER reaction. A mutant of Bacillus caldotenax UvrA (Bca UvrA) was generated by substituting a glycine for eleven of the highly conserved amino acids within the C-terminal zinc finger. The mutant protein, ZnG UvrA, was purified and characterized. Our results demonstrate that the mutant ZnG UvrA can bind dsDNA, but has lost its damage-specific dsDNA binding, and cannot complement for in vivo UV resistance.EXPERIMENTAL PROCEDURESProteins—The C-terminal ABC ATPase domain (CABC fragment) of uvrABca was amplified using the following primers (5′-GCG ACC GGA TCC ATG CTG GCC GCG GAC TAT TTG and 5′-GAG AGA GCG GCC GCT TAC GCC TTC ACC GCT TCA TAT TG) and the Pfu turbo DNA polymerase (Stratagene). The CABC fragment includes nucleotides 1643 to the end of the uvrABca gene; thus this fragment includes the linker region and the C-terminal ABC motifs, amino acids 549-952. The PCR fragment was cloned into a Topo cloning vector, pCRBII-TOPO, then transferred into the pGEX4T1 vector by digestion with BamH1 and NotI to create pGEX4T1-Wt CABC.Construction of the ZnG deletion mutant of pGEX-CABC was performed with the QuikChange site-directed mutagenesis kit from Stratagene using pGEX4T1-Wt CABC as template, primers (5′-CAT GGC GAT GGC ATC ATC GGT GTC CCG TGC GAA GTG TGC CAC and 5′-GTG GCA CAC TTC GCA CGG GAC ACC GAT GAT GCC ATC GCC ATG) and Pfu turbo DNA polymerase. An alignment of the zinc finger region showing which amino acids are deleted is depicted in Fig. 1.To place the ZnG mutation into the full-length uvrA gene, the BseR1 and NcoI fragment from pGEX4T1-ZnG CABC was removed, gel-purified, and ligated into the BseR1/NcoI (New England Biolabs) double-digested, artic phosphatase-treated pTYB1-Wt uvrABca vector, thus completing the construction of pTYB1-ZnG uvrABca.The plasmid coding for GST-ZnF, pGEX6p1-ZnF was created by PCR amplification of nucleotides 2002-2463 by Pfu turbo DNA polymerase primers (5′-ATA GGG ATC CGG CGA GCA CCG CGA CAT TC and 5′-ACA CGC GGC CGC GCC GAG CTT CAT ATA ACC). The PCR fragment was cloned into a Topo cloning vector, pCRBII-TOPO, then transferred into the pGEX6p1 vector by digestion with BamH1 and NotI to create pGEX6p-ZnF.The inserts of all vectors were sequenced. Upon re-sequencing the pTYB1-Wt uvrABca expression vector, we discovered several sequence variations from the original Bca UvrA sequence deposited in Entrez, accession number AAK29748. There were a total of fourteen amino acid changes; however none reside within any conserved sequence elements, (sequence conservation defined as 70% or more identity among 24 UvrA homologues). The majority of the variations, 13 of the 14, were in the poorly conserved linker region. Of all the variations, only linker region substitution Phe600 to Thr600 is unique to the new Bca UvrA sequence. The remaining amino acid changes exist in at least one or more UvrA homologues. The Wt UvrA, ZnG UvrA, Wt GST-CABC, and ZnG GST-CABC proteins used in this study share these fourteen amino acid substitutions.Expression and Purification of the Proteins-Bca Wt UvrA, Bca ZnG UvrA, Bca Wt UvrB, Bca Δβ-hairpin UvrB, and Thermatoga maritima Wt UvrC (Tma UvrC)—All proteins were expressed in BL21 (DE3) RIL cells (Stratagene) and purified using the T7 IMPACT™ system (New England Biolabs) by standard procedures. The GST-containing proteins were expressed in BL21 (DE3) RIL cells (Stratagene) and purified using glutathione-Sepharose 4B resin (GE Healthcare). The GST-containing proteins were eluted from the column with reduced glutathione (10 mm) in 20 mm Tris-HCl, pH 8, 0.5 m NaCl, 0.1 mm EDTA, 5 mm DTT, and 0.25% Triton X-100, then dialyzed into storage buffer. UvrA, UvrB, and the GST fusion proteins were maintained at -20 °C in storage buffer (50 mm Tris-HCl, pH 7.5, 500 mm KCl, 0.1 mm EDTA, and 50% glycerol) until use.DNA Substrates—DNA substrates were synthesized by Sigma-Genosys. The DNA sequence of the 50-mer double-stranded substrate containing a single internal fluorescein (FldT) adduct was: F2650 5′-GAC TAC GTA CTG TTA CGG CTC CAT C[FldT]C TAC CGC AAT CAG GCC AGA TCT GC-3′ while the non-damaged complementary bottom strand was NDB, 5′-GCA GAT CTG GCC TGA TTG CGG TAG CGA TGG AGC CGT AAC AGT ACG TAG TC-3′. The non-damaged strand, NDT, has the same sequence as F2650 except it contains a dT at the position of FldT. The DNA was 5′ end-labeled using T4 polynucleotide kinase and [γ-32P]ATP (3000 Ci/mmol, Amersham Biosciences) according to standard procedures. The reaction was terminated by the addition of EDTA (20 mm), and the enzyme was heat-denatured by incubation for 10 min at 65 °C. Unincorporated radioactive nucleotides were removed by gel filtration chromatography (Biospin-6, Bio-Rad). The labeled oligonucleotide was annealed with equimolar amounts of the complementary oligonucleotide.When referring to the oligonucleotide duplexes, the strand listed first is the one that is 5′ end-labeled. The double-stranded character of the oligonucleotide duplex was analyzed on a native 10% polyacrylamide gel.UV Survival Assay—WP2 uvrA- trp- cells (Mol Tox, Inc., Boone, NC) were incubated with 50 ng of pT7pol26 plasmid (Gentaur, Belgium) and 50 ng of pTYB1(New England Biolabs), pTYB1-Wt UvrABca or pTYB1-ZnG UvrABca for 10 min. Wt WP2 cells (MolTox, Inc.) were incubated with 50 ng of pT7pol26 and 50 ng of pTYB1 or pTYB1-ZnG for 10 min. Transformations were carried out in cuvettes with a 0.2-cm electrode gap using a Gene Pulser II (Bio-Rad) electroporator with peak discharge at 2.4 kV, resistance set at 100 ohms and capacitance set to 25 microfarads. Immediately following transformation, cells were transferred into 250 μl of SOC broth. After 1 h of shaking at 37 °C, the entire culture was spread onto LB plates containing 100 μg/ml ampicillin and 50 μg/ml kanamycin. Individual colonies were selected and grown to an A600 of ∼1.0. Then the cell culture was diluted 2-fold, and the proteins were induced by addition of IPTG (0.1 mm) for 1 h at 37 °C. This concentration of IPTG gave low, but detectable levels of UvrA protein. In duplicate, three serial dilutions of each sample of culture (100 μl) were spread onto LB plates containing 50 μg/ml kanamycin and 100 μg/ml ampicillin and UV-irradiated. The appropriate UV dose was calculated by measuring the fluency from an 8-watt 254-nm germicidal lamp using a 254-nm UVX Radiometer (UVP Inc.). Serial dilutions of unirradiated cultures were also plated and used to determine the plating efficiency of each transformant. The number of colonies obtained after 20 h of incubation at 37 °C was recorded, and the percent survival calculated from the plating efficiency of the nonirradiated controls. Two or three independent experiments were performed for each sample. The mean survival of two-three independent experiments is plotted as a function of UV fluence.Incision Assay—Prior to initiation of the incision assay, the UvrABC proteins were heated to 65 °C for 10 min. The 5′-end-labeled duplex DNA (2 nm, F2650/NDB) was treated with UvrABC (20 nm Wt or ZnG Bca UvrA, 100 nm Bca UvrB, and 50 nm Tma UvrC) in 20 μl of UvrABC buffer (50 mm Tris-HCl, pH 7.5, 50 mm KCl, 10 mm MgCl2, 1 mm ATP, and 5 mm DTT) at 55 °C for the indicated time. For those reactions containing supercoiled undamaged plasmid DNA, varying concentrations of pUC19 DNA (New England Biolabs) were included as labeled in the figure legend. The reactions were terminated by addition of EDTA (20 mm). Ten percent of the reaction was removed, denatured with formamide and heated to 85 °C for 5 min. Incision products were resolved on a 10% denaturing polyacrylamide gel, and electrophoresis was performed at 325 V in Tris borate-EDTA buffer (89 mm Tris, 89 mm boric acid, and 2 mm EDTA) for 40 min. Gels were dried and exposed to a PhosphorImager screen (Molecular Dynamics) overnight. The percent of the DNA incised was calculated using the Molecular Dynamics software, ImageQuant to determine the band intensities within each lane. The percentage of DNA incised is reported as the mean ± S.D. (n = 3).Electrophoretic Mobility Shift Assay (EMSA)—For UvrA·DNA EMSAs, the binding reactions were performed with duplexed DNA substrate (2 nm) and protein (concentrations as indicated in the figure legend) in 20-μl reaction buffer (50 mm Tris-HCl, pH 7.5, 100 mm KCl, 10 mm MgCl2, 1 mm ATP, 5 mm DTT, and 1 μm bovine serum albumin) for 15 min at 37 °C or 55 °C (as indicated in the figure legend). The reactions were loaded onto a 3.5% native polyacrylamide gel (29:1; acrylamide:bis). For EMSAs containing ATP and magnesium, the gels and running buffer contained 44.5 mm Tris pH 8.3, 44.5 mm boric acid, and 1 mm EDTA, 1 mm ATP and 10 mm MgCl2. For EMSAs without ATP and magnesium, the gels and running buffer contained 44.5 mm Tris, 44.5 mm boric acid, and 1 mm EDTA. Electrophoresis was carried out for 1 h at 100 V with the gel rigs at 4 °C. The gels were dried and exposed to a phosphorimager screen. The percent of DNA bound in the various protein-DNA complexes was calculated based on the total radioactivity in the lane. The percentage is reported as the mean ± S.D. (n = 3). Data are reported as the fraction of DNA-bound, including higher order multimers, versus protein concentration and fitted by nonlinear regression analysis (17Schofield M.J. Lilley D.M. White M.F. Biochemistry. 1998; 37: 7733-7740Crossref PubMed Scopus (42) Google Scholar).For the UvrA·UvrB·DNA EMSAs, the enzymes were preheated to 65 °C for 10 min prior to initiation of the reactions and UvrA·UvrB reaction mixtures. Binding reactions were performed with 2 nm F2650/NDB and Wt UvrA (20 nm) or ZnG UvrA (20 nm) and Wt UvrB (100 nm) in 20 μl of reaction buffer (50 mm Tris-HCl, pH 7.5, 100 mm KCl, 10 mm MgCl2, 1 mm ATP, 5 mm DTT, and 1 μm bovine serum albumin) for 30 min at 55 °C. For those lanes that contain additional plasmid DNA, a 100-fold molar excess of pUC DNA (molar excess relative to the concentration of oligonucleotide in base pairs) was added prior to adding the oligonucleotide to the reactions. The reactions were loaded onto a 4% native polyacrylamide gel (29:1, acrylamide:bis) and subjected to electrophoresis at 100 V for 1 h, 4 °C. The gels and buffers contained 44.5 mm Tris, 44.5 mm boric acid and 1 mm EDTA, 1 mm ATP, and 10 mm MgCl2.ATP Hydrolysis Assay—The conversion of ATP to ADP by the UvrAB system was monitored using a coupled enzyme assay system consisting of pyruvate kinase and lactic dehydrogenase to couple the hydrolysis of ATP to the oxidation of NADH (ϵ340 nm = 6220 m-1 cm-1). ATP (Roche Applied Science) was added to a final concentration of 1 mm in a 100-μl reaction mixture containing 50 mm Tris-HCl, pH 7.5, 55 mm KCl, 4 mm MgCl2, 1 mm DTT, 12.6 units/ml l-lactic dehydrogenase (Sigma), 10 units/ml pyruvate kinase (Sigma), 2 mm phosphoenol pyruvate (Roche Applied Science), 0.15 mm NADH (Roche Applied Science), 50 nm Bca UvrA (Wt or mutants), and 100 nm Bca UvrB. The Bca proteins were preheated to 65 °C for 10 min prior to initiation of the reactions. Each protein was assayed in the absence of DNA as well as in the presence of supercoiled pUC19 (SC DNA, 10 ng/μl) or UV-irradiated pUC18 DNA (UV DNA, 10 ng/μl) substrate. The UV-damaged DNA was prepared by exposure of 1 μg/μl pUC18 plasmid DNA to 200 J/m2 for 1 min. The rate of hydrolysis was calculated from the linear change in absorbance at 340 nm at either 37 or 55 °C over a 20-30-min period, using a Beckman DU-640 spectrophotometer. In addition, the rates were blank corrected for the oxidation of NADH (+ATP) in the absence of additional proteins. For ATPase reactions at 37 °C, the proteins were not preheated prior to the assay. For those reactions at 55 °C, the data are reported as the mean rate (M/min) ± S.D. (n = 3 or 4).Creation of the Zinc Finger Structural Model—The C-terminal zinc finger domain was modeled based on an alignment with the coordinates from the solved structure of Ydj1. Ydj1, the Saccharomyces cerevisiae dnaJ homologue, is a protein chaperone involved in the regulation of Hsp90 and Hsp70 (18Caplan A.J. Douglas M.G. J. Cell Biol. 1991; 114: 609-621Crossref PubMed Scopus (210) Google Scholar, 19Kimura Y. Yahara I. Lindquist S. Science. 1995; 268: 1362-1365Crossref PubMed Scopus (218) Google Scholar, 20Ziegelhoffer T. Lopez-Buesa P. Craig E.A. J. Biol. Chem. 1995; 270: 10412-10419Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The structure of Ydj1 was solved in complex with its peptide substrate (PDB code 1NLT) (21Li J. Qian X. Sha B. Structure (Camb.). 2003; 11: 1475-1483Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The Ydj1 zinc finger structure was selected as the template to model the UvrA zinc finger as they share a conserved CXXCXGXG sequence, which is common to this zinc finger protein family, referred to as DNAJ_CXXCXGXG (Pfam record PF00684, Ref. 22Bateman A. Coin L. Durbin R. Finn R.D. Hollich V. Griffiths-Jones S. Khanna A. Marshall M. Moxon S. Sonnhammer E.L. Studholme D.J. Yeats C. Eddy S.R. Nucleic Acids Res. 2004; 32: D138-D141Crossref PubMed Google Scholar). In the DnaJ family of proteins, CXXCXGXG is repeated four times. In the C-terminal domain of UvrA, the sequence CXXCXGX(R/K) is repeated twice. The lysine-containing consensus is also found among the DnaJ proteins. From the 20 DnaJ protein sequences that form the Pfam seed alignment for Pfam record PF00684, the CXXCXGX(R/K) is found 30% of the time within the fourth DnaJ repeat. The sequence alignment of Ydj1 and several UvrA proteins used to develop the structural model for the UvrA C-terminal zinc finger is shown in Fig. 1.Development of a Structural Model for the ABC ATPase within the C-terminal Domain of UvrA, Residues 603-713 and 801-935—A comparative model was developed using structural (DALI/FSSP, VAST) and sequence (T-Coffee, ClustalW) alignments of solved ABC transporter proteins including the MalK ATPase subunit of maltose ABC transporter (PDB code 1G29, Ref. 23Diederichs K. Diez J. Greller G. Muller C. Breed J. Schnell C. Vonrhein C. Boos W. Welte W. EMBO J. 2000; 19: 5951-5961Crossref PubMed Scopus (273) Google Scholar), the MJ0796 ATP-binding cassette protein (PDB code 1L2T, Ref. 24Smith P.C. Karpowich N. Millen L. Moody J.E. Rosen J. Thomas P.J. Hunt J.F. Mol. Cell. 2002; 10: 139-149Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar), and the ATP binding subunit of histidine permease (PDB code 1B0U, Ref. 25Hung L.W. Wang I.X. Nikaido K. Liu P.Q. Ames G.F. Kim S.H. Nature. 1998; 396: 703-707Crossref PubMed Scopus (614) Google Scholar). These structures were selected because they were solved at high resolution (1.9, 1.7, and 1.5 Å, respectively) and shared the highest sequence similarity with UvrA. Coordinates from all three solved structures contributed to the model; however, the majority was from 1B0U, which shares 32% sequence identity with UvrA in the ABC transporter domain regions. The RMS deviation between the solved ABC transporter structures used to develop the UvrA model was 3.7-3.9 Å (backbone Cα). The ATP was placed in the model UvrA structure based on the position of ATP within the histidine permease solved structure (PDB code 1B0U). The distances between the ATP atoms and atoms of the ABC ATPase-conserved motifs (Walker A, Q-loop, ABC signature, Walker B, and His-loop) were optimized. The model was manually edited using the Accelrys InsightII Homology Module Software. The final model was subjected to minimization and short (200 ps) molecular dynamics runs to resolve discontinuities with CHARMm. The RMS deviation between the final UvrA ABC transporter monomer model and the 1B0U PDB structure is 3.2 Å.A model for the UvrA C-terminal dimer was constructed using the solved Rad50 dimer structure (PDB codes: 1F2U, 1F2T) as a model template for the interactions between the two monomeric C-terminal ABC transporter domains of UvrA. Each UvrA monomer was superimposed onto each of the Rad50 monomers so that the UvrA dimer structural interactions would simulate that of the Rad50 dimer organization. ATP atoms and the conserved atoms belonging to the ABC transporter motifs were used for the superposition of each of the ABC monomeric units with the Rad50 monomer to create UvrA C-terminal dimer model coordinates. The RMS deviation between the Rad50 dimer and UvrA C-terminal dimer is 4.8 Å.RESULTSDesign and Construction of ZnG UvrA—Prior research on the UvrA C-terminal zinc finger focused on disrupting the zinc-coordinating amino acids. Specifically, substitution of E. coli UvrA C-terminal zinc finger cysteine, C763F, produced a protein that was unable to bind dsDNA; therefore, this led to the hypothesis that the C-terminal zinc finger region of UvrA was responsible for DNA binding (16Wang J. Mueller K.L. Grossman L. J. Biol. Chem. 1994; 269: 10771-10775Abstract Full Text PDF PubMed Google Scholar). Visse et al. (14Visse R. de Ruijter M. Ubbink M. Brandsma J.A. van de Putte P. Mutat. Res. 1993; 294: 263-274Crossref PubMed Scopus (22) Google Scholar) also mutated one of the C-terminal cysteines and noted that the structural stability of the mutant protein was impaired. The zinc ion plays an important role in the structural architecture of zinc fingers and mutagenesis of the zinc-anchoring cysteines probably disrupts the global fold of the C-terminal ABC ATPase domain.Unlike many other zinc finger DNA-binding proteins (13Matthews J.M. Sunde M. IUBMB Life. 2002; 54: 351-355Crossref PubMed Scopus (240) Google Scholar), UvrA is not a sequence-specific DNA-binding protein. Furthermore, close inspection of the amino acids within and around the C-terminal zinc finger reveals that the conserved amino acids tend to be hydrophobic, whereas only a few are positively charged residues, which may be available to interact with the DNA phosphate backbone. For these reasons, we re-investigated the role of the C-terminal zinc finger domain of UvrA.A multiple sequence alignment of the UvrA C-terminal zinc finger region is displayed in Fig. 1A. Inspection of the sequences reveals a common motif: C(D/E)XCXGXGX3(I/V)EMXFLPDX4 C(D/E)XCXG. Note that there are conserved glycines after each set of cysteines. The dnaJ family of proteins possess a similar zinc finger signature; however dnaJ proteins have 4 repeats of CX2CXGX(G/K). The structure of the yeast DnaJ homolog, Ydj1, has been solved (PDB 1NLT, Ref. 21Li J. Qian X. Sha B. Structure (Camb.). 2003; 11: 1475-1483Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), and its model is shown in Fig. 1B. A model of the UvrA C-terminal zinc finger was created based on the structure of the ZnII domain of Ydj1 because it contains 22 amino acids between the conserved cysteines, similar to the UvrA spacing of 19 amino acids, (Fig. 1C). Based on the structural model and sequence alignments, a deletion mutant of UvrA was created. This deletion mutant replaced eleven of the amino acids within the C-terminal finger with a single glycine residue (Fig. 1A).In Vivo Complementation and Survival—In vivo complementation and UV survival studies were conducted in the WP2 uvrA- strain. Transformation of WP2 uvrA- cells with plasmids encoding T7 polymerase and Bca Wt UvrA increases the cells UV survival by more than 382-fold, pTYB1 versus pTYB1-Wt UvrA, when exposed to 5 J/m2 of UV. These results indicate that at 37 °C, Bca Wt UvrA can complement the E. coli system. In contrast, transformation of pTYB1-ZnG UvrA versus pTYB1 only confers a 43-fold increase in UV survival following 5 J/m2 of UV. The UV survival differences between Wt UvrA and ZnG UvrA transformed samples varied between 8.6-12.4-fold. Clearly, the ZnG UvrA protein is defective at some step in the NER reaction because it provides a lower level of UV protection than Wt Bca UvrA.Wild-type WP2 cells were also included in the UV survival analysis. Even though Wt Bca UvrA provided significant protection from UV, it did not provide the same level of defense as endogenous E. coli UvrA, compare Wt WP2 with Wt UvrA in Fig. 2. Bca UvrA was 3-10-fold less effective after UV than endogenous E. coli UvrA. The ZnG UvrA containing vector was also transformed into the Wt WP2 cells to determine if ZnG UvrA would be a dominant negative mutation. The Wt WP2 cells expressing ZnG UvrA do not display a greater sensitivity to UV than cells transformed with empty vector (Fig. 2). Therefore, ZnG UvrA does not exert a dominant negative affect on endogenous E. coli UvrA.FIGURE 2UV survival of E. coli WP2 strains. WP2 (trp-, uvrA-) cells were transformed with pT7pol26, a plasmid encoding an IPTG-inducible T7 polymerase, and pTYB1 or pTYB1-Wt UvrA or pTYB1-ZnG UvrA. In addition, WP2 (trp-) cells with endogenous UvrA were transformed with pT7pol26 and pTYB1 or pTYB1-ZnG UvrA. After individual colonies were selected and grown to an A600 of ∼1.0, the cell culture was diluted 2-fold, and the proteins were induced by addition of IPTG (0.1 mm) for 1 h at 3}, number={36}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Croteau, Deborah L. and DellaVecchia, Matthew J. and Wang, Hong and Bienstock, Rachelle J. and Melton, Mark A. and Van Houten, Bennett}, year={2006}, month={Jul}, pages={26370–26381} }
 @article{wang_dellavecchia_skorvaga_croteau_erie_van houten_2006, title={UvrB Domain 4, an Autoinhibitory Gate for Regulation of DNA Binding and ATPase Activity}, volume={281}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/jbc.M601476200}, DOI={10.1074/jbc.M601476200}, abstractNote={UvrB, a central DNA damage recognition protein in bacterial nucleotide excision repair, has weak affinity for DNA, and its ATPase activity is activated by UvrA and damaged DNA. Regulation of DNA binding and ATP hydrolysis by UvrB is poorly understood. Using atomic force microscopy and biochemical assays, we found that truncation of domain 4 of Bacillus caldotenax UvrB (UvrBDelta4) leads to multiple changes in protein function. Protein dimerization decreases with an approximately 8-fold increase of the equilibrium dissociation constant and an increase in DNA binding. Loss of domain 4 causes the DNA binding mode of UvrB to change from dimer to monomer, and affinity increases with the apparent dissociation constants on nondamaged and damaged single-stranded DNA decreasing 22- and 14-fold, respectively. ATPase activity by UvrBDelta4 increases 14- and 9-fold with and without single-stranded DNA, respectively, and UvrBDelta4 supports UvrA-independent damage-specific incision by Cho on a bubble DNA substrate. We propose that other than its previously discovered role in regulating protein-protein interactions, domain 4 is an autoinhibitory domain regulating the DNA binding and ATPase activities of UvrB.}, number={22}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Wang, Hong and DellaVecchia, Matthew J. and Skorvaga, Milan and Croteau, Deborah L. and Erie, Dorothy A. and Van Houten, Bennett}, year={2006}, month={Apr}, pages={15227–15237} }
 @article{truglio_rhau_croteau_wang_skorvaga_karakas_dellavecchia_wang_van houten_kisker_2005, title={Structural insights into the first incision reaction during nucleotide excision repair}, volume={24}, ISSN={0261-4189 1460-2075}, url={http://dx.doi.org/10.1038/sj.emboj.7600568}, DOI={10.1038/sj.emboj.7600568}, abstractNote={Article3 February 2005free access Structural insights into the first incision reaction during nucleotide excision repair James J Truglio James J Truglio Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY, USA Search for more papers by this author Benjamin Rhau Benjamin Rhau Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY, USA Search for more papers by this author Deborah L Croteau Deborah L Croteau Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA Search for more papers by this author Liqun Wang Liqun Wang Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY, USA Search for more papers by this author Milan Skorvaga Milan Skorvaga Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia Search for more papers by this author Erkan Karakas Erkan Karakas Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY, USA Search for more papers by this author Matthew J DellaVecchia Matthew J DellaVecchia Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA Search for more papers by this author Hong Wang Hong Wang Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA Search for more papers by this author Bennett Van Houten Bennett Van Houten Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA Search for more papers by this author Caroline Kisker Corresponding Author Caroline Kisker Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY, USA Search for more papers by this author James J Truglio James J Truglio Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY, USA Search for more papers by this author Benjamin Rhau Benjamin Rhau Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY, USA Search for more papers by this author Deborah L Croteau Deborah L Croteau Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA Search for more papers by this author Liqun Wang Liqun Wang Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY, USA Search for more papers by this author Milan Skorvaga Milan Skorvaga Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia Search for more papers by this author Erkan Karakas Erkan Karakas Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY, USA Search for more papers by this author Matthew J DellaVecchia Matthew J DellaVecchia Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA Search for more papers by this author Hong Wang Hong Wang Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA Search for more papers by this author Bennett Van Houten Bennett Van Houten Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA Search for more papers by this author Caroline Kisker Corresponding Author Caroline Kisker Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY, USA Search for more papers by this author Author Information James J Truglio1, Benjamin Rhau1, Deborah L Croteau2, Liqun Wang1, Milan Skorvaga2,3, Erkan Karakas1, Matthew J DellaVecchia2, Hong Wang2, Bennett Van Houten2 and Caroline Kisker 1 1Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY, USA 2Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA 3Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia *Corresponding author. Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794-5115, USA. Tel.: +1 631 632 1465; Fax: +1 631 632 1555; E-mail: [email protected] The EMBO Journal (2005)24:885-894https://doi.org/10.1038/sj.emboj.7600568 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Nucleotide excision repair is a highly conserved DNA repair mechanism present in all kingdoms of life. The incision reaction is a critical step for damage removal and is accomplished by the UvrC protein in eubacteria. No structural information is so far available for the 3′ incision reaction. Here we report the crystal structure of the N-terminal catalytic domain of UvrC at 1.5 Å resolution, which catalyzes the 3′ incision reaction and shares homology with the catalytic domain of the GIY-YIG family of intron-encoded homing endonucleases. The structure reveals a patch of highly conserved residues surrounding a catalytic magnesium-water cluster, suggesting that the metal binding site is an essential feature of UvrC and all GIY-YIG endonuclease domains. Structural and biochemical data strongly suggest that the N-terminal endonuclease domain of UvrC utilizes a novel one-metal mechanism to cleave the phosphodiester bond. Introduction Nucleotide excision repair (NER) stands apart from other DNA repair mechanisms available to the cell in its ability to recognize a broad range of structurally unrelated DNA damages (Van Houten, 1990; Friedberg et al, 1995; Lloyd and Van Houten, 1995; Sancar, 1996; Goosen and Moolenaar, 2001) including carcinogenic cyclobutane pyrimidine dimers induced by UV radiation, benzo[a]pyrene-guanine adducts caused by smoking and burning of fossil fuels, and guanine-cisplatinum adducts formed during cancer chemotherapy (Sancar, 1994). The strategy employed by NER is the same in all three kingdoms of life. NER in prokaryotes was one of the first repair mechanisms discovered (Boyce and Howard-Flanders, 1964; Setlow and Carrier, 1964) and is mediated by the UvrA, UvrB and UvrC proteins. These three proteins recognize and cleave damaged DNA in an ATP-dependent multistep reaction. UvrA is involved in damage recognition and either forms a heterotrimeric (UvrA2UvrB) (reviewed in Theis et al, 2000) or heterotetrameric (UvrA2UvrB2) (Verhoeven et al, 2002) complex with UvrB. This complex is believed to scan the DNA helix for conformational perturbations induced by DNA lesions (Theis et al, 2000). After the damage has been identified, UvrA dissociates from the protein–DNA complex, leaving UvrB bound to the DNA (Orren and Sancar, 1990) forming a stable preincision complex (Theis et al, 2000; Skorvaga et al, 2002). UvrC binds to this complex and mediates the incision three or four nucleotides 3′ to the damaged site, followed by a second incision seven nucleotides 5′ to the damaged site (Sancar and Rupp, 1983; Lin and Sancar, 1992a, 1992b; Verhoeven et al, 2000). UvrD (helicase II) and DNA polymerase I (polI) are required for turnover of the UvrABC proteins (Caron et al, 1985; Husain et al, 1985). UvrD removes both UvrC and the oligonucleotide containing the lesion, while UvrB remains bound to the gapped DNA until it is displaced by DNA polI (Orren et al, 1992). The reaction is completed by DNA ligase, which closes the nicked DNA. This multistep process of DNA recognition and repair ensures a high degree of discrimination between the damaged and nondamaged strand. Site-directed mutagenesis and sequence alignments have shown that UvrC catalyzes both the 3′ and 5′ incisions and each of these incisions is performed by a distinct catalytic site that can be inactivated independently (Lin and Sancar, 1992b; Verhoeven et al, 2000). The domain responsible for 3′ incision is located in the N-terminal half of the molecule and consists of approximately the first hundred residues. This domain shares limited homology with a small module found in members of the GIY-YIG endonuclease family (Aravind et al, 1999). Also included in the N-terminal half is a region that interacts with the C-terminal domain of UvrB (Aravind et al, 1999). The 5′ catalytic domain, which is distantly related to Escherichia coli endonuclease V, is located in the C-terminal half of the protein along with two helix-hairpin-helix motifs employed in DNA binding (Aravind et al, 1999). After recruitment to the UvrB:DNA preincision complex, UvrC first catalyzes cleavage of the DNA on the 3′ side of the lesion (Verhoeven et al, 2000). This incision requires the interaction between the C-terminal domain of UvrB and the homologous UvrB binding domain of UvrC (Moolenaar et al, 1995, 1998a), which is not required for 5′ incision (Moolenaar et al, 1995). In order to obtain a better understanding of the 3′ incision event, we have solved the crystal structure of the N-terminal endonuclease domain of UvrC from two different thermophilic organisms, Bacillus caldotenax and Thermotoga maritima, at 2.0 and 1.5 Å resolution, respectively. This domain shares structural and sequence similarity to the catalytic domain found in I-TevI, a GIY-YIG homing endonuclease (Van Roey et al, 2002). I-TevI is one of at least 60 known GIY-YIG endonuclease family members that are present in bacteriophage T4, bacteria, archaea, algal chloroplasts and mitochondria, and fungal mitochondria. Members of this family are characterized by a 70–100 residues long module containing a conserved GIY-(X9–11)-YIG motif. We have identified a patch of highly conserved residues on the surface of the N-terminal domain of UvrC to which a single divalent cation is bound. The residues that form the metal binding pocket are conserved throughout all GIY-YIG endonucleases, suggesting the site to be a common feature of all family members. We mutated seven amino acids within the conserved patch and analyzed whether full-length UvrC was still able to incise damaged DNA, in the complete UvrABC reaction. Combined with the structural data, the results indicate that the conserved patch is the active site and the bound divalent cation is the catalytic metal. Based on our data, we propose that UvrC uses a novel one-metal mechanism to catalyze cleavage of the fourth or fifth phosphodiester bond 3′ to the DNA lesion. Results Crystal structure of the N-terminal endonuclease domain of UvrC The N-terminal catalytic domain of UvrC was initially cloned from B. caldotenax (UvrCN-Bca; residues 1–98) and its structure was solved by multiwavelength anomalous diffraction (MAD) (Table I). The protein crystallized in space group C2 and contained four molecules in the asymmetric unit arranged as a tetramer with C4 symmetry. Each of the four subunits has nearly identical conformations, with an average root mean square (r.m.s.) deviation of 0.62 Å for the Cα atoms of residues 8–94. The structure was refined at 2.0 Å resolution to an R-factor of 0.203 and Rfree of 0.252 (Table I). Residues 95–98 of subunit A, 1–8 and 95–98 of subunit B, 95–98 of subunit C, as well as 1 and 96–98 of subunit D are disordered. Table 1. Crystallographic statistics Data set Native (Bca) SeMet peak (Bca) SeMet inflection (Bca) SeMet remote (Bca) Native (Tma) Mn2+ bound native (Tma) Mg2+ bound native (Tma) Resolution (Å) 2.0 2.0 2.0 2.0 1.8 1.5 1.8 Wavelength (Å) 1.0 0.9794 0.9798 0.9538 1.1 1.1 1.1 Unique reflections 29 029 56 919 56 982 56 947 16 312 27 459 16 305 〈I〉/〈σI〉 37.2 (5.58) 31.1 (2.74) 31.6 (3.36) 31.6 (3.34) 47.5 (4.88) 40.5 (4.57) 35.8 (4.02) Completeness (%) 100.0 (100.0) 98.2 (97.6) 98.2 (97.6) 98.2 (97.6) 99.9 (100.0) 98.4 99.7 Rsym 0.07 (0.43) 0.07 (0.70) 0.06 (0.53) 0.06 (0.53) 0.06 (0.60) 0.04 (0.31) 0.06 (0.52) Phasing to 3.0 Å FOM (from SOLVE) 0.78 (0.68) Map correlation 0.45 Mean phase difference (deg) 55.0 (60.7) Rcryst (Rfree) 0.203 (0.252) 0.185 (0.199) 0.167 (0.185) 0.175 (0.202) r.m.s. deviation bond lengths (Å) 0.014 0.017 0.013 0.017 r.m.s. deviation bond angles (deg) 1.5 1.4 1.5 1.5 Mean B-factor 36.1 26.0 24.8 21.8 Ramachandran Statistics 94.3/4.8/0.9/0.0 94.9/3.8/1.3/0.0 94.9/5.1/0.0/0.0 96.2/2.6/1.3/0.0 Tma and Bca refer to T. maritima and B. caldotenax UvrC, respectively. Rsym=∑hkl∑i∣Ii–〈I〉∣/∑hkl∑i〈I〉, where Ii is the ith measurement and 〈I〉 is the weighted mean of all measurements of I. 〈I〉/〈σI〉 indicates the average of the intensity divided by its average standard deviation. Numbers in parentheses refer to the respective highest resolution data shell in each data set. Rcryst=∑∣∣Fo∣−∣Fc∣∣/∑∣Fo∣, where Fo and Fc are the observed and calculated structure factor amplitudes. Rfree is same as Rcryst for 5% of the data randomly omitted from the refinement. The map correlation coefficient describes the correlation between the electron density map calculated from the final model and the map corresponding to the experimental set of phases, averaged over all grid points. The mean phase difference is the mean differences between the initial phases calculated from SOLVE and phases calculated from the final wild-type model. Ramachandran statistics indicates the fraction of residues in the most favored, additionally allowed, generously allowed and disallowed regions of the Ramachandran diagram, as defined by the program PROCHECK (Laskowski et al, 1993). The corresponding domain was also cloned from T. maritima (UvrCN-Tma; residues 1–97) and crystallized in space group P43212 containing one molecule in the asymmetric unit. Crystals were soaked with manganese or magnesium chloride. The structure of UvrCN-Tma bound to manganese was solved by molecular replacement using the UvrCN-Bca monomer as a search model (see Materials and methods). This structure was refined at 1.5 Å resolution to an R-factor of 0.167 and Rfree of 0.185 (Table I), and consists of residues 1–89. The structure of magnesium-bound UvrCN-Tma was solved using difference Fourier methods (Table I). For the remainder of the discussion, residue numbering will correspond to UvrC from T. maritima, unless specified otherwise. The B. caldotenax and T. maritima structures have similar conformations with an r.m.s. deviation of 1.55 Å for 88 Cα atoms. Both have an αββααβα(α) topology with the C-terminal α-helix (α5) not present in UvrCN-Tma. The core of the molecule is a three-stranded β-sheet, which is flanked by the first three helices on one side and helix 4 on the other (Figure 1). Helices α1, α2 and α4 run approximately parallel to the β-sheet, while helix α3 is positioned almost perpendicular to these secondary structural elements, making contact only with the bottom edge of the sheet. In UvrCN-Bca helix α3 also interacts with the C-terminus, connecting the two helical halves of the protein across the β-sheet. Figure 1.: Stereo view of the N-terminal endonuclease domain of UvrCN-Tma. The central β-sheet (β1–β3) is shown in yellow and the surrounding helices (α1–α4) in green. Conserved residues are shown in ball-and-stick representation and the Mg2+ ion as a magenta sphere. The N- and C-termini of the domain are indicated. This figure and Figures 2A, 4 and 8 were generated with the programs MOLSCRIPT (Kraulis, 1991) and RASTER3D (Merritt and Murphy, 1994). Download figure Download PowerPoint Comparison to the catalytic domain of I-TevI A search using DALI (Holm and Sander, 1995), a network service for comparing three-dimensional protein structures (http://www.ebi.ac.uk/dali), identified the catalytic domain of I-TevI (Van Roey et al, 2002) (PDB codes 1LN0 and 1MK0) as the only structure with a fold similar to the N-terminal domain of UvrC (Z-score of 7.8). I-TevI is a member of the GIY-YIG family of homing endonucleases, which in turn belong to the larger GIY-YIG superfamily that includes UvrC. The only entity in common among all superfamily members is the domain presented here. It is a small, 70–100 residues module, containing a conserved GIY-(X9–11)-YIG motif (Van Roey et al, 2002) (Gly 17, Val 18, Tyr 19–Tyr 29, Ile 30, Gly 31), four invariant residues (Gly 31, Arg 39, Glu 76, Asn 88) and two highly conserved residues (Tyr 19, Tyr 29) (Figure 1). These amino acids are all located in proximity to each other and form, in part, a highly conserved surface. The catalytic domains of UvrC and I-TevI have likely diverged long ago as reflected by their low sequence identity of only 15% (Figure 2B). UvrCN-Tma superimposes onto the catalytic domain of I-TevI with an r.m.s. deviation of 2.2 Å for 60 out of 89 possible Cα atoms. However, there are notable differences in secondary and tertiary structure (Figure 2A). UvrCN-Tma/Bca contains an additional helix, α1, as compared to I-TevI. The function of α1 in UvrC is undoubtedly structural, as the residues that form this helix are not conserved among UvrCs, and sequence alignments show that like I-TevI, certain UvrC proteins lack this N-terminal helix (Figure 3B). Secondly, the fragment in UvrCN-Tma/Bca spanning α2 and β3, which includes α3, is not structurally conserved compared to I-TevI. Regardless of this dissimilarity, the position of Ile 54 from UvrCN-Tma (Leu 56 in UvrCBca) is conserved and superimposes well with Leu 45 in I-TevI (Figure 2A and B). This residue is structurally important and stabilizes the hydrophobic core of the domain. Lastly, UvrCN-Bca contains an additional helix, α5, at its C-terminus that is replaced by a loop region in both I-TevI and UvrCN-Tma (Figure 2A and B). Figure 2.Structural comparison of the two 3′ endonuclease domains from UvrC and the analogous domain in I-TevI. (A) Following their superposition, the three proteins were separated and displayed side by side: UvrCN-Tma (left), UvrCN-Bca (middle) and I-TevI (right). Selected residues are shown in ball-and-stick representation; hydrogen bonds are indicated by dotted lines. (B) Structure-based sequence alignment of UvrC from T. maritima and B. caldotenax, and I-TevI. Secondary structure elements are indicated above and below the sequence alignment corresponding to UvrC and I-TevI, respectively. The blue lines below the secondary structure elements indicate large regions of structural dissimilarity. Uppercase letters indicate residues that align structurally, while lowercase letters indicate residues that are not structurally aligned. Selected, structurally aligned residues are highlighted in red. Residues in a similar position, but not structurally aligned are highlighted in blue. Numbers above the sequence alignment correspond to residue numbering in UvrCN-Tma and UvrCN-Bca with numbering from UvrCN-Bca in superscript. Numbers below the alignment relate to residue numbering in I-TevI. Download figure Download PowerPoint Figure 3.Sequence conservation of the 3′ endonuclease domain of UvrC. (A) Two different views of the N-terminal domain in surface representation. The right view is rotated 90° relative to the left view around the vertical axis. Only the most conserved residues with solvent-accessible side chains are labeled with the addition of Phe 73. Color-coding is according to conservation (green: strictly conserved; blue: highly conserved; black: moderately conserved). (B) Sequence alignment of the N-terminal GIY-YIG domain from four selected UvrCs. The sequences are from T. maritima (gi:8134799), B. caldotenax, E. coli (gi:38704033) and Deinococcus radiodurans (gi:6116772). Secondary structure elements are indicated above the sequence and refer to the structure of UvrCN-Tma. They are color-coded according to Figure 1. Conserved residues are color-coded as in (A). The conserved GIY-(X9–11)-YIG sequence for which the domain was named is indicated by two red lines above the sequences. Part A and Figure 6 were generated with the programs SPOCK (Christopher and Baldwin, 1998) and RASTER3D. Download figure Download PowerPoint Both UvrCN-Tma/Bca and I-TevI contain a strictly conserved glutamate, arginine and asparagine, and a pair of highly conserved tyrosine residues (Tyr 19 and Tyr 29 in UvrCTma; Tyr 6 and Tyr 17 in I-TevI) (Figure 2A). The third tyrosine, Tyr 43 in UvrCN-Tma, corresponds to His 31 in I-TevI (Figure 2A and B). Sequence alignments reveal that this residue is either a tyrosine or a histidine in all UvrCs (Figure 3B) and all other GIY-YIG family members. Both Tyr 43 of UvrCN-Tma and His 31 of I-TevI form a hydrogen bond to Tyr 19 and Tyr 6, respectively (Figure 2A). His 31 of I-TevI in turn forms a hydrogen bond to His 40, which occupies the position of Val 51 in UvrC. In place of a hydrogen bond, Val 51 forms van der Waals interactions with Tyr 43. The conserved surface Mapping the sequence conservation of UvrC proteins from different organisms onto the surface reveals a conserved patch of amino acids (Figure 3A and B). Six strictly conserved residues are located in the center on one side of the surface: Tyr 19, Tyr 29, Lys 32, Arg 39, Glu 76 and Asn 88. These strictly conserved amino acids are surrounded by a number of highly conserved residues: Tyr 43, Phe 73 and Ile 80. The conserved residues form a shallow, concave surface with dimensions of 16 Å × 15 Å and 5 Å deep, which could easily accommodate double-stranded DNA. This is in agreement with biochemical data, which have shown that the 3′ endonuclease site only recognizes double-stranded DNA as a substrate and DNA containing an unpaired region of more than eight nucleotides overlapping the 3′ incision site is not incised (Zou and Van Houten, 1999). The cleft is sufficient for nuclease activity; however, the isolated domain does not bind to DNA (data not shown) and requires either the UvrB interacting domain and/or the C-terminal helix-hairpin-helix DNA binding domain of UvrC for catalysis to occur. The divalent cation Phosphodiester bonds, although thermodynamically labile, require large activation energies for cleavage at physiological pH (Galburt and Stoddard, 2002). This is mainly due to the negative charge of the phosphoryl group at this pH, which repels potential attacking nucleophiles. Three chemical entities are required to catalyze efficiently the cleavage of the phosphodiester bond: a general base to position and activate the nucleophile (usually a water molecule) for inline attack of the 5′ phosphate, a general acid to protonate the 3′ leaving group and a Lewis acid to stabilize the pentacovalent phosphoanion transition state. A number of different strategies to satisfy these requirements have evolved. A common feature of most nucleases is the use of one, two or even three divalent cations in the active site to lower the free energy of the transition state (Galburt and Stoddard, 2002). Metal ions can decrease the pKa of coordinating water molecules, resulting in a bound hydroxide, which could take the role of either a nucleophile or a general base. Alternatively, a metal-coordinated water molecule can be acidic and has the potential to serve as the general acid necessary to protonate the 3′ OH leaving group. However, the most substantial reduction in free energy derives from the metal's ability to stabilize the negative charge of the phosphoanion transition state (Galburt and Stoddard, 2002). No divalent cation was observed in the B. caldotenax structure after soaking and cocrystallization attempts. This is presumably due to the crystallization conditions, which contained high salt concentrations at low pH (5.4). In addition, there were protein–protein interactions in proximity to the only strictly conserved negatively charged amino acid, Glu 76, where metal binding was predicted to occur. Fortunately, crystals of UvrCN-Tma grew in PEG 8000 at high pH (8.5), a condition more suitable for soaking experiments. Additionally, Glu 76 was completely solvent accessible with no nearby protein–protein interactions. These crystals were briefly soaked with either MnCl2 or MgCl2 and the resulting structures provided similar results: a single divalent cation coordinated by Glu 76 and five well-ordered water molecules in an octahedral arrangement (Figure 4). These structures provide the first view of the metal and its exact coordination geometry for any member of the GIY-YIG superfamily and thus structural insight into the catalytic mechanism. The bound manganese was clearly identified in anomalous density maps, while omit maps revealed the octahedral arrangement of the chelating water molecules (Figure 4). The refined density for the water molecules in both structures is unambiguous with B-factors ranging from 17.1 to 23.6 Å2 for the manganese structure and 21.1 to 28.1 Å2 for the magnesium structure. The B-factors for the manganese and magnesium ion are 18.8 and 25.6 Å2, respectively. The manganese structure superimposes onto the magnesium structure with an r.m.s. deviation of 0.1 Å, and the two metal-water clusters superimpose with an r.m.s. deviation of 0.1 Å as well. Figure 4.Stereo view of the active site of the 3′ endonuclease domain. The metal ion is shown as a magenta sphere and the five surrounding water molecules as red spheres. Hydrogen bonds are shown as dotted lines. A simulated annealing omit map omitting the magnesium-water cluster, Glu 76, Arg 39 and Tyr 29 is shown at 1σ (blue, transparent) and an anomalous map is shown at 7σ (green cage). Residues in close proximity to the metal ion are shown in ball-and-stick representation. Download figure Download PowerPoint Although Glu 76 is the only protein residue bound directly to the metal, the water molecules coordinating to the metal form additional contacts to the protein (Figure 4). One of the waters forms hydrogen bonds to both the hydroxyl group of Tyr 29 and the main-chain carbonyl of Ile 30. A second water forms a hydrogen bond to the carboxylate of Glu 76, while a third water forms a hydrogen bond to the backbone amide of Lys 32. Residues forming the binding pocket for the metal-water cluster are highly conserved, suggesting the metal binding site to be a conserved feature of all GIY-YIG family members. Mutational analysis of the conserved surface Based on our structural data, we generated mutations of the highly conserved surface residues Y29A, Y29F, K32A, R39A, F73A, F73E, E76A, I80A, I80E and N88A in full-length UvrCTma and tested these mutant proteins in an incision assay using a defined substrate and the two other NER proteins UvrABca and UvrBBca (Figure 5). In addition, we verified that all of the mutants were able to bind and crosslink to double-stranded DNA. Electrophoretic mobility shift assays were performed using wild-type and mutant UvrC proteins (in the absence of UvrA or UvrB) with a 40 bp duplex containing a centrally located (+)-trans-BPDE adduct. Crosslinking was performed using wild-type and mutant UvrC proteins (in the absence of UvrA or UvrB) with 50 bp duplex containing a site-specific arylazido-modified photoaffinity reagent (as described in DellaVecchia et al, 2004) (data not shown). Figure 5.Incision activity of T. maritima UvrC mutants. The 5′ (A) or 3′ (B) end-labeled 50-mer double-stranded DNA substrates containing a centrally located fluorescein (FldT) were incubated with 20 nM UvrABca, 100 nM UvrBBca and 12.5 nM of the indicated UvrCTma protein for 30 min at 55°C in reaction buffer. The reactions were terminated with stop buffer, and the incision products were analyzed on a 10% denaturing polyacrylamide gel. (C) Comparison of the incision activity using the 3′-labeled substrate (gray bars) and 5′-labeled substrate (black bars) and the indicated UvrC proteins. Data are reported as the mean±the standard deviation of the mean of at least four incision assays per UvrC protein. Download figure Download PowerPoint Mutation of the sole metal ligand, Glu 76, to alanine renders UvrC unable to mediate either the 3′ or 5′ incision (Figure 5). UvrC from Tma, like E. coli UvrC, cannot achieve 5′ incision without prior 3′ incision, and thus inactivation of the 3′ nuclease active site inhibits the 5′ nuclease activity. Other inactive mutants were N88A, Y29A, Y29F, R39A and I80E. Mutant I80A showed an approximately 50% reduction in activity. Mutation of Lys 32 to alanine resulted in a protein that was 25–30% less active than wild-type UvrC and mutation of Phe 73 to either alanine or glutamate resulted in an enzyme with wild-type UvrC activity (Figure 5). The Y19F and Y43F mutants did not overexpress and could not be studied. In addition to the full-length mutants, we generated mutations in the isolated N-terminal domain (Y19F, Y29F, Y43F and N88A) and determined the structures of these mutants to inspect whether structural changes within the mutants lead to the inactivation of the enzyme (see Supplementary Table I). Discussion In this study, we have crystallized and solved the s}, number={5}, journal={The EMBO Journal}, publisher={Wiley}, author={Truglio, James J and Rhau, Benjamin and Croteau, Deborah L and Wang, Liqun and Skorvaga, Milan and Karakas, Erkan and DellaVecchia, Matthew J and Wang, Hong and Van Houten, Bennett and Kisker, Caroline}, year={2005}, month={Feb}, pages={885–894} }
 @article{truglio_rhau_croteau_wang_skorvaga_karakas_dellavecchia_wang_van houten_kisker_2005, title={Structural insights into the first incision reaction during nucleotide excision repair}, volume={24}, number={5}, journal={The EMBO journal}, publisher={EMBO Press}, author={Truglio, James J and Rhau, Benjamin and Croteau, Deborah L and Wang, Liqun and Skorvaga, Milan and Karakas, Erkan and DellaVecchia, Matthew J and Wang, Hong and Van Houten, Bennett and Kisker, Caroline}, year={2005}, pages={885–894} }
 @article{van houten_croteau_dellavecchia_wang_kisker_2005, title={‘Close-fitting sleeves’: DNA damage recognition by the UvrABC nuclease system}, volume={577}, ISSN={0027-5107}, url={http://dx.doi.org/10.1016/j.mrfmmm.2005.03.013}, DOI={10.1016/j.mrfmmm.2005.03.013}, abstractNote={DNA damage recognition represents a long-standing problem in the field of protein-DNA interactions. This article reviews our current knowledge of how damage recognition is achieved in bacterial nucleotide excision repair through the concerted action of the UvrA, UvrB, and UvrC proteins.}, number={1-2}, journal={Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis}, publisher={Elsevier BV}, author={Van Houten, Bennett and Croteau, Deborah L. and DellaVecchia, Matthew J. and Wang, Hong and Kisker, Caroline}, year={2005}, month={Sep}, pages={92–117} }
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 @article{xue_ratcliff_wang_davis-searles_gray_erie_redinbo_2002, title={A minimal exonuclease domain of WRN forms a hexamer on DNA and possesses both 3'-5'exonuclease and 5'-protruding strand endonuclease activities}, volume={41}, number={9}, journal={Biochemistry}, publisher={ACS Publications}, author={Xue, Yu and Ratcliff, Glenn C and Wang, Hong and Davis-Searles, Paula R and Gray, Matthew D and Erie, Dorothy A and Redinbo, Matthew R}, year={2002}, pages={2901–2912} }
 @article{drotschmann_hall_shcherbakova_wang_erie_brownewell_kool_kunkel_2002, title={DNA binding properties of the yeast Msh2-Msh6 and Mlh1-Pms1 heterodimers}, volume={383}, number={6}, journal={Biological chemistry}, author={Drotschmann, Karin and Hall, Mark C and Shcherbakova, Polina V and Wang, Hong and Erie, Dorothy A and Brownewell, Floyd R and Kool, Eric T and Kunkel, Thomas A}, year={2002}, pages={969–975} }
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