@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{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. 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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. 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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. 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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{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. 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. Telomeres are nucleoprotein structures that prevent the degradation or fusion of the ends of linear chromosomes, which are threatened by at least seven distinct DNA damage response (DDR) pathways (1Palm W. de Lange T. How shelterin protects mammalian telomeres.Annu. Rev. Genet. 2008; 42: 301-334Crossref PubMed Scopus (1344) Google Scholar, 2Muraki K. Nyhan K. Han L. Murnane J.P. Mechanisms of telomere loss and their consequences for chromosome instability.Front. Oncol. 2012; 2: 135Crossref PubMed Google Scholar, 3de Lange T. Shelterin-mediated telomere protection.Annu. Rev. Genet. 2018; 52: 223-247Crossref PubMed Scopus (280) Google Scholar). 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However, since TIN2 itself does not directly bind to DNA and instead serves as a "mediator/enhancer" for shelterin and telomerase activities, defining TIN2's distinct function at the molecular level has been challenging. The bottleneck for studying TIN2 lies in the fact that results from bulk biochemical assays do not fully reveal the heterogeneity and dynamics of the protein–protein and protein–DNA interactions. Furthermore, cell-based assays only provide information on the outcomes from downstream effectors after the knocking down of TIN2 that also removes TRF1 and TRF2 from telomeres. These approaches do not allow us to investigate the molecular structures and dynamics in which TIN2 directly participates. In vivo, the amount of TIN2 is sufficient for binding every TRF1 and TRF2 molecule (44Takai K.K. Hooper S. Blackwood S. Gandhi R. de Lange T. In vivo stoichiometry of shelterin components.J. Biol. Chem. 2010; 285: 1457-1467Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), while TPP1 and POT1 are ~10-fold less than TRF1 and TIN2. Thus, it is important to study the DNA-binding properties of TRF1-TIN2 complexes. To fill this important knowledge gap, we applied complementary single-molecule imaging platforms, including atomic force microscopy (AFM) (45Yang Y. Wang H. Erie D.A. Quantitative characterization of biomolecular assemblies and interactions using atomic force microscopy.Methods. 2003; 29: 175-187Crossref PubMed Scopus (88) Google Scholar, 46Wang H. Nora G.J. Ghodke H. Opresko P.L. Single molecule studies of physiologically relevant telomeric tails reveal POT1 mechanism for promoting G-quadruplex unfolding.J. Biol. Chem. 2011; 286: 7479-7489Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 47Kaur P. Wu D. Lin J. Countryman P. Bradford K.C. Erie D.A. Riehn R. Opresko P.L. Wang H. Enhanced electrostatic force microscopy reveals higher-order DNA looping mediated by the telomeric protein TRF2.Sci. Rep. 2016; 6: 20513Crossref PubMed Scopus (20) Google Scholar), total internal reflection fluorescence microscopy (TIRFM) (48Erie D.A. Weninger K.R. Single molecule studies of DNA mismatch repair.DNA Repair. 2014; 20: 71-81Crossref PubMed Scopus (46) Google Scholar), and the DNA tightrope assay to monitor TRF1-TIN2-mediated DNA compaction and DNA-DNA bridging (49Lin J. Countryman P. Chen H. Pan H. Fan Y. Jiang Y. Kaur P. Miao W. Gurgel G. You C. Piehler J. Kad N.M. Riehn R. Opresko P.L. Smith S. 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 (18) Google Scholar, 50Countryman P. Fan Y. Gorthi A. Pan H. Strickland J. Kaur P. Wang X. Lin J. Lei X. White C. You C. Wirth N. Tessmer I. Piehler J. Riehn R. et al.Cohesin SA2 is a sequence-independent DNA-binding protein that recognizes DNA replication and repair intermediates.J. Biol. Chem. 2018; 293: 1054-1069Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 51Pan H. Jin M. Ghadiyaram A. Kaur P. Miller H.E. Ta H.M. Liu M. Fan Y. Mahn C. Gorthi A. You C. Piehler J. Riehn R. Bishop A.J.R. Tao Y.J. 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). Through using DNA substrates on different length scales (6 and 270 TAAGGG repeats), these imaging platforms provide complementary results demonstrating that both TIN2S and TIN2L facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. In some cases, TRF1-TIN2 is capable of mediating the bridging of multiple copies of telomeric DNA fragments. Importantly, our results demonstrate that TIN2 protects the disassembly of TRF1-TIN2-mediated DNA-DNA bridging by Tankyrase 1. In addition, the N-terminal domain of TPP1 inhibits TRF1-TIN2-mediated DNA-DNA bridging. In summary, this study uncovered the unique biophysical function of TIN2 as a telomeric architectural protein, acting together with TRF1 to mediate interactions between distant telomeric sequences. Tankyrase 1 and TPP1 regulate TRF1-TIN2-mediated DNA-DNA bridging. Furthermore, this work establishes a unique combination of single-molecule imaging platforms for future examination of TIN2 disease variants and provides a new direction for investigating molecular mechanisms underlying diverse TIN2 functions. A previous study suggested that TIN2 modulates the bridging of telomeric DNA by TRF1 (31Kim S.H. Han S. You Y.H. Chen D.J. Campisi J. The human telomere-associated protein TIN2 stimulates interactions between telomeric DNA tracts in vitro.EMBO Rep. 2003; 4: 685-691Crossref PubMed Scopus (42) Google Scholar). However, the bulk biochemical assays using short telomeric DNA (six telomeric repeats) did not provide information regarding the structure and dynamics of the TRF1-TIN2-DNA complex. To investigate the molecular function of TIN2, we applied AFM imaging to investigate how TIN2 affects the telomeric DNA-DNA pairing mediated by TRF1 at the single-molecule level on longer telomeric DNA substrates (270 TTAGGG repeats). We purified TRF1 (Fig. S1A) and obtained TIN2S (1–354 amino acids, 39.4 kDa) and TIN2L (1–451 amino acids, 50.0 kDa) proteins purified from insect cells (Fig. 1A and Fig. S1D). Previously, we established an AFM imaging-based calibration method to investigate the oligomeric states and protein–protein interactions by correlating AFM volumes of proteins and their molecular weights (45Yang Y. Wang H. Erie D.A. Quantitative characterization of biomolecular assemblies and interactions using atomic force microscopy.Methods. 2003; 29: 175-187Crossref PubMed Scopus (88) Google Scholar, 47Kaur P. Wu D. Lin J. Countryman P. Bradford K.C. Erie D.A. Riehn R. Opresko P.L. Wang H. Enhanced electrostatic force microscopy reveals higher-order DNA looping mediated by the telomeric protein TRF2.Sci. Rep. 2016; 6: 20513Crossref PubMed Scopus (20) Google Scholar, 52Wang H. Yang Y. Erie D.A. Characterization of protein-protein interactions using atomic force microscopy.in: Schuck P. Protein Interactions Biophysical approaches for the Study of Complex Reversible Systems. Springer Science+Business Media, LLC, Berlin, Germany2007: 39-78Crossref Google Scholar). AFM volumes of TRF1 alone in solution showed two distinct peaks, which were consistent with TRF1 monomers (51 KDa) and dimers (102 KDa, Fig. S1B). In addition, based on the population of TRF1 under the monomer and dimer peaks (53Wang H. DellaVecchia M.J. Skorvaga M. Croteau D.L. Erie D.A. Van Houten B. UvrB domain 4, an autoinhibitory gate for regulation of DNA binding and ATPase activity.J. Biol. Chem. 2006; 281: 15227-15237Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), the estimated TRF1 dimer equilibrium dissociation constant (Kd) is 18.4 nM (Fig. S1C). Meanwhile, AFM volumes of purified TIN2S at 41.3 nm3 (±28.3 nm3) and TIN2L at 41.9 nm3 (±12.8 nm3) were consistent with the notion that TIN2 does not interact with itself (23Kim S.H. Kaminker P. Campisi J. TIN2, a new regulator of telomere length in human cells.Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (418) Google Scholar), and TIN2 exists in a monomeric state in solution (Fig. S1D). Furthermore, we conducted size-exclusive chromatography using TRF1 and TIN2S and confirmed the presence of TRF1 dimers, TIN2 monomers, as well as the interaction between TRF1 and TIN2S in solution (Fig. S2). To further validate the activities of TIN2, we used electrophoresis mobility shift assays (EMSAs) to verify the interaction of TIN2 with TRF1 on a double-stranded telomeric DNA substrate (48 bp containing three TTAGGG repeats, Fig. S3, A–C). Consistent with previous studies (23Kim S.H. Kaminker P. Campisi J. TIN2, a new regulator of telomere length in human cells.Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (418) Google Scholar), EMSA experiments showed that TIN2S and TIN2L did not directly bind to telomeric dsDNA (Fig. S3A). Both TRF1-TIN2S and TRF1-TIN2L induced a clear supershift of the telomeric DNA substrate compared with TRF1 alone (Complex III in Fig. S3, B and C), indicating the formation of stable TRF1-TIN2-telomeric DNA complexes. Next, to study TRF1-TIN2 DNA binding at the single-molecule level, we used the linear DNA substrate (5.4 kb) that contains 1.6 kb (270 TTAGGG) telomeric repeats in the middle region that is 35%–50% from DNA ends (T270 DNA, Experimental procedures, Fig. 1A) (21Lin J. Countryman P. Buncher N. Kaur P. E L. Zhang Y. Gibson G. You C. Watkins S.C. Piehler J. Opresko P.L. Kad N.M. Wang H. 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 (44) Google Scholar, 49Lin J. Countryman P. Chen H. Pan H. Fan Y. Jiang Y. Kaur P. Miao W. Gurgel G. You C. Piehler J. Kad N.M. Riehn R. Opresko P.L. Smith S. 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 (18) Google Scholar). Previously, AFM and electron microscopy imaging–based studies established that TRF1 specifically binds to the telomeric region and mediates DNA-DNA pairing (21Lin J. Countryman P. Buncher N. Kaur P. E L. Zhang Y. Gibson G. You C. Watkins S.C. Piehler J. Opresko P.L. Kad N.M. Wang H. 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 (44) Google Scholar, 22Bianchi A. Stansel R.M. Fairall L. Griffith J.D. Rhodes D. de Lange T. TRF1 binds a bipartite telomeric site with extreme spatial flexibility.EMBO J. 1999; 18: 5735-5744Crossref PubMed Scopus (163) Google Scholar, 49Lin J. Countryman P. Chen H. Pan H. Fan Y. Jiang Y. Kaur P. Miao W. Gurgel G. You C. Piehler J. Kad N.M. Riehn R. Opresko P.L. Smith S. 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 (18) Google Scholar). To study the function of TIN2, we preincubated TRF1 without or with TIN2 (either TIN2S or TIN2L), followed by the addition}, 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{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"]}, DOI={10.1093/nar/gkab1142}, abstractNote={Abstract}, number={22}, journal={NUCLEIC ACIDS RESEARCH}, 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}, 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{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}, number={1}, journal={EPIGENETICS & CHROMATIN}, 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.}, 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{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{green_huang_pan_han_lim_2018, title={Optical Temperature Sensing With Infrared Excited Upconversion Nanoparticles}, volume={6}, ISSN={["2296-2646"]}, DOI={10.3389/fchem.2018.00416}, abstractNote={Upconversion Nanoparticles (UCNPs) enable direct measurement of the local temperature with high temporal and thermal resolution and sensitivity. Current studies focusing on small animals and cellular systems, based on continuous wave (CW) infrared excitation sources, typically lead to localized thermal heating. However, the effects of upconversion bioimaging at the molecular scale, where higher infrared intensities under a tightly focused excitation beam, coupled with pulsed excitation to provide higher peak powers, is not well understood. We report on the feasibility of 800 and 980 nm excited UCNPs in thermal sensing under pulsed excitation. The UCNPs report temperature ratiometrically with sensitivities in the 1 × 10−4 K−1 range under both excitation wavelengths. Our optical measurements show a ln(I525/I545) vs. 1/T dependence for both 800 nm and 980 nm excitations. Despite widespread evidence promoting the benefits of 800 nm over 980 nm CW excitation in avoiding thermal heating in biological imaging, in contrary, we find that given the pulsed laser intensities appropriate for single particle imaging, at both 800 and 980 nm, that there is no significant local heating in air and in water. Finally, in order to confirm the applicability of infrared imaging at excitation intensities compatible with single nanoparticle tracking, DNA tightropes were exposed to pulsed infrared excitations at 800 and 980 nm. Our results show no appreciable change in the viability of DNA over time when exposed to either wavelengths. Our studies provide evidence for the feasibility of exploring protein-DNA interactions at the single molecule scale, using UCNPs as a reporter.}, journal={FRONTIERS IN CHEMISTRY}, author={Green, Kory and Huang, Kai and Pan, Hai and Han, Gang and Lim, Shuang Fang}, year={2018}, month={Sep} }
 @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={Abstract 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{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={Abstract 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{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} }