@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}, 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{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}, 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.}, 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}, 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. 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). Human telomeres contain ~2–20 kb of TTAGGG repeats and a G-rich 3′ overhang of ~50–400 nt in length (1Palm W. de Lange T. How shelterin protects mammalian telomeres.Annu. Rev. Genet. 2008; 42: 301-334Crossref PubMed Scopus (1344) Google Scholar, 4Wright W.E. Tesmer V.M. Huffman K.E. Levene S.D. Shay J.W. Normal human chromosomes have long G-rich telomeric overhangs at one end.Genes Dev. 1997; 11: 2801-2809Crossref PubMed Scopus (572) Google Scholar). In humans, a specialized six-protein shelterin complex consisting of TRF1, TRF2, RAP1, TIN2, TPP1, and POT1 binds specifically to the unique sequence and structure at telomeres to protect chromosome ends. Prevention of telomeres from being falsely recognized as double-strand DNA breaks and regulation of DNA repair protein access depend on the biochemical activities of shelterin proteins and their collaborative actions with other proteins involved in the genome maintenance pathways (5Cech T.R. Beginning to understand the end of the chromosome.Cell. 2004; 116: 273-279Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 6Songyang Z. Liu D. Inside the mammalian telomere interactome: Regulation and regulatory activities of telomeres.Crit. Rev. Eukaryot. Gene Expr. 2006; 16: 103-118Crossref PubMed Scopus (38) Google Scholar, 7Verdun R.E. Karlseder J. Replication and protection of telomeres.Nature. 2007; 447: 924-931Crossref PubMed Scopus (370) Google Scholar, 8Canudas S. Houghtaling B.R. Kim J.Y. Dynek J.N. Chang W.G. Smith S. Protein requirements for sister telomere association in human cells.EMBO J. 2007; 26: 4867-4878Crossref PubMed Scopus (80) Google Scholar, 9Giraud-Panis M.J. Pisano S. Poulet A. Le Du M.H. Gilson E. Structural identity of telomeric complexes.FEBS Lett. 2010; 584: 3785-3799Crossref PubMed Scopus (36) Google Scholar). Extensive telomere shortening or dramatic telomere loss due to DNA damage causes telomere deprotection, which triggers cell senescence and aging-related pathologies (10d'Adda di Fagagna F. Reaper P.M. Clay-Farrace L. Fiegler H. Carr P. Von Zglinicki T. Saretzki G. Carter N.P. Jackson S.P. A DNA damage checkpoint response in telomere-initiated senescence.Nature. 2003; 426: 194-198Crossref PubMed Scopus (1979) Google Scholar, 11Jaskelioff M. Muller F.L. Paik J.H. Thomas E. Jiang S. Adams A.C. Sahin E. Kost-Alimova M. Protopopov A. Cadinanos J. Horner J.W. Maratos-Flier E. Depinho R.A. Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice.Nature. 2011; 469: 102-106Crossref PubMed Scopus (556) Google Scholar). The main protein–protein and protein–DNA interactions at telomeres have been investigated using crystallography, biochemical assays, yeast two-hybrid systems, coimmunoprecipitation, as well as visualization of shelterin subcomponents in vitro and in vivo using fluorescence imaging (3de Lange T. Shelterin-mediated telomere protection.Annu. Rev. Genet. 2018; 52: 223-247Crossref PubMed Scopus (280) Google Scholar). Among shelterin components, both TRF1 and TRF2 specifically recognize double-stranded telomeric DNA through the Myb/SANT domain facilitated by homodimerization through the TRFH domain (12Broccoli D. Chong L. Oelmann S. Fernald A.A. Marziliano N. van Steensel B. Kipling D. Le Beau M.M. de Lange T. Comparison of the human and mouse genes encoding the telomeric protein, TRF1: Chromosomal localization, expression and conserved protein domains.Hum. Mol. Genet. 1997; 6: 69-76Crossref PubMed Scopus (80) Google Scholar, 13Broccoli D. Smogorzewska A. Chong L. de Lange T. Human telomeres contain two distinct Myb-related proteins, TRF1 and TRF2.Nat. Genet. 1997; 17: 231-235Crossref PubMed Scopus (741) Google Scholar). However, TRF1 and TRF2 display distinct DNA-binding properties and functions. TRF1 and TRF2 contain an acidic and a basic domain, respectively, at their N-termini (14Poulet A. Pisano S. Faivre-Moskalenko C. Pei B. Tauran Y. Haftek-Terreau Z. Brunet F. Le Bihan Y.V. Ledu M.H. Montel F. Hugo N. Amiard S. Argoul F. Chaboud A. Gilson E. et al.The N-terminal domains of TRF1 and TRF2 regulate their ability to condense telomeric DNA.Nucleic Acids Res. 2012; 40: 2566-2576Crossref PubMed Scopus (55) Google Scholar). TRF2 prevents Mre11/Rad50/Nbs1-dependent ATM kinase signaling, classical nonhomologous end-joining (NHEJ), as well as alternative nonhomologous end-joining (alt-NHEJ) pathways at telomeres. These distinct functions of TRF2 are believed to be mediated through its activities in promoting and stabilizing T-loops, in which the 3′ single-stranded overhang invades the upstream double-stranded telomeric region (15Griffith J.D. Comeau L. Rosenfield S. Stansel R.M. Bianchi A. Moss H. de Lange T. Mammalian telomeres end in a large duplex loop.Cell. 1999; 97: 503-514Abstract Full Text Full Text PDF PubMed Scopus (1886) Google Scholar, 16Doksani Y. Wu J.Y. de Lange T. Zhuang X. Super-resolution fluorescence imaging of telomeres reveals TRF2-dependent T-loop formation.Cell. 2013; 155: 345-356Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 17Benarroch-Popivker D. Pisano S. Mendez-Bermudez A. Lototska L. Kaur P. Bauwens S. Djerbi N. Latrick C.M. Fraisier V. Pei B. Gay A. Jaune E. Foucher K. Cherfils-Vicini J. Aeby E. 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 (85) Google Scholar, 18Van Ly D. Low R.R.J. Frolich S. Bartolec T.K. Kafer G.R. Pickett H.A. Gaus K. Cesare A.J. Telomere loop dynamics in chromosome end protection.Mol. Cell. 2018; 71: 510-525.e516Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In comparison, TRF1 represses telomere fragility by preventing DNA replication fork stalling at telomeres (19Sfeir A. Kosiyatrakul S.T. Hockemeyer D. MacRae S.L. Karlseder J. Schildkraut C.L. de Lange T. Mammalian telomeres resemble fragile sites and require TRF1 for efficient replication.Cell. 2009; 138: 90-103Abstract Full Text Full Text PDF PubMed Scopus (685) Google Scholar) and promotes parallel pairing of telomeric DNA tracts (20Griffith J. Bianchi A. de Lange T. TRF1 promotes parallel pairing of telomeric tracts in vitro.J. Mol. Biol. 1998; 278: 79-88Crossref PubMed Scopus (123) Google Scholar, 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). A flexible domain in TRF1 enables the two Myb domains in the TRF1 dimer to interact with DNA independently and to mediate looping of telomeric DNA (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). TIN2 itself does not have binding affinity to either double-stranded or single-stranded DNA (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). However, it is a core shelterin component that bridges double-stranded (TRF1 and TRF2) and single-stranded telomeric DNA-binding proteins (TPP1-POT1) (24Kim S.H. Beausejour C. Davalos A.R. Kaminker P. Heo S.J. Campisi J. TIN2 mediates functions of TRF2 at human telomeres.J. Biol. Chem. 2004; 279: 43799-43804Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 25Houghtaling B.R. Cuttonaro L. Chang W. Smith S. A dynamic molecular link between the telomere length regulator TRF1 and the chromosome end protector TRF2.Curr. Biol. 2004; 14: 1621-1631Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 26Kim S.H. Davalos A.R. Heo S.J. Rodier F. Zou Y. Beausejour C. Kaminker P. Yannone S.M. Campisi J. Telomere dysfunction and cell survival: Roles for distinct TIN2-containing complexes.J. Cell Biol. 2008; 181: 447-460Crossref PubMed Scopus (44) Google Scholar, 27O'Connor M.S. Safari A. Xin H. Liu D. Songyang Z. A critical role for TPP1 and TIN2 interaction in high-order telomeric complex assembly.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 11874-11879Crossref PubMed Scopus (181) Google Scholar, 28Ye J.Z. Hockemeyer D. Krutchinsky A.N. Loayza D. Hooper S.M. Chait B.T. de Lange T. POT1-interacting protein PIP1: A telomere length regulator that recruits POT1 to the TIN2/TRF1 complex.Genes Dev. 2004; 18: 1649-1654Crossref PubMed Scopus (340) Google Scholar). Crystal structures revealed the interfaces between TRF1-TIN2, TRF2-TIN2, and TPP1-TIN2 (29Hu C. Rai R. Huang C. Broton C. Long J. Xu Y. Xue J. Lei M. Chang S. Chen Y. Structural and functional analyses of the mammalian TIN2-TPP1-TRF2 telomeric complex.Cell Res. 2017; 27: 1485-1502Crossref PubMed Scopus (50) Google Scholar, 30Chen Y. Yang Y. van Overbeek M. Donigian J.R. Baciu P. de Lange T. Lei M. A shared docking motif in TRF1 and TRF2 used for differential recruitment of telomeric proteins.Science. 2008; 319: 1092-1096Crossref PubMed Scopus (187) Google Scholar). TIN2 stabilizes both TRF1 and TRF2 at telomeres (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, 32Ye J.Z. Donigian J.R. van Overbeek M. Loayza D. Luo Y. Krutchinsky A.N. Chait B.T. de Lange T. TIN2 binds TRF1 and TRF2 simultaneously and stabilizes the TRF2 complex on telomeres.J. Biol. Chem. 2004; 279: 47264-47271Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). The loss of the TRF1 or TRF2-binding domains in TIN2 triggers a DNA damage response (33Takai K.K. Kibe T. Donigian J.R. Frescas D. de Lange T. Telomere protection by TPP1/POT1 requires tethering to TIN2.Mol. Cell. 2011; 44: 647-659Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Binding of TPP1 to TIN2 is required for POT1-mediated telomere protection (34Frescas D. de Lange T. Binding of TPP1 protein to TIN2 protein is required for POT1a,b protein-mediated telomere protection.J. Biol. Chem. 2014; 289: 24180-24187Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). As an integral component of the “TIN2/TPP1/POT1 processivity complex,” TIN2 functions together with TPP1/POT1 to stimulate telomerase processivity (35Pike A.M. Strong M.A. Ouyang J.P.T. Greider C.W. TIN2 functions with TPP1/POT1 to stimulate telomerase processivity.Mol. Cell. Biol. 2019; 39e00593-18Crossref PubMed Scopus (22) Google Scholar). Furthermore, TIN2 directly interacts with the cohesin subunit SA1 and plays a key role in a distinct SA1-TRF1-TIN2-mediated sister telomere cohesion pathway that is largely independent of the cohesin ring subunits (8Canudas S. Houghtaling B.R. Kim J.Y. Dynek J.N. Chang W.G. Smith S. Protein requirements for sister telomere association in human cells.EMBO J. 2007; 26: 4867-4878Crossref PubMed Scopus (80) Google Scholar, 36Canudas S. Smith S. Differential regulation of telomere and centromere cohesion by the Scc3 homologues SA1 and SA2, respectively, in human cells.J. Cell Biol. 2009; 187: 165-173Crossref PubMed Scopus (122) Google Scholar). Binding of TRF1-TIN2 to telomeres is regulated by the poly(ADP-ribose) polymerase Tankyrase 1 (37Smith S. Giriat I. Schmitt A. de Lange T. Tankyrase, a poly(ADP-ribose) polymerase at human telomeres.Science. 1998; 282: 1484-1487Crossref PubMed Scopus (891) Google Scholar). ADP-ribosylation of TRF1 by Tankyrase 1 reduces its binding to telomeric DNA in vitro, and the depletion of Tankyrase 1 using siRNA leads to mitotic arrest and persistent telomere cohesion that can be rescued by depletion of TIN2 (8Canudas S. Houghtaling B.R. Kim J.Y. Dynek J.N. Chang W.G. Smith S. Protein requirements for sister telomere association in human cells.EMBO J. 2007; 26: 4867-4878Crossref PubMed Scopus (80) Google Scholar, 36Canudas S. Smith S. Differential regulation of telomere and centromere cohesion by the Scc3 homologues SA1 and SA2, respectively, in human cells.J. Cell Biol. 2009; 187: 165-173Crossref PubMed Scopus (122) Google Scholar, 38Dynek J.N. Smith S. Resolution of sister telomere association is required for progression through mitosis.Science. 2004; 304: 97-100Crossref PubMed Scopus (216) Google Scholar). Three distinct TIN2 isoforms have been identified in human cell lines (35Pike A.M. Strong M.A. Ouyang J.P.T. Greider C.W. TIN2 functions with TPP1/POT1 to stimulate telomerase processivity.Mol. Cell. Biol. 2019; 39e00593-18Crossref PubMed Scopus (22) Google Scholar, 39Kaminker P.G. Kim S.H. Desprez P.Y. Campisi J. A novel form of the telomere-associated protein TIN2 localizes to the nuclear matrix.Cell Cycle. 2009; 8: 931-939Crossref PubMed Scopus (35) Google Scholar, 40Smith S. The long and short of it: A new isoform of TIN2 in the nuclear matrix.Cell Cycle. 2009; 8: 797-798Crossref PubMed Scopus (2) Google Scholar) that include TIN2S (354 AAs), TIN2L (451 AAs), and TIN2M (TIN2 medium, 420 AAs). TIN2S, TIN2L, and TIN2M share the same TRF1, TRF2, and TPP1-binding domains and localize to telomeres (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, 35Pike A.M. Strong M.A. Ouyang J.P.T. Greider C.W. TIN2 functions with TPP1/POT1 to stimulate telomerase processivity.Mol. Cell. Biol. 2019; 39e00593-18Crossref PubMed Scopus (22) Google Scholar, 39Kaminker P.G. Kim S.H. Desprez P.Y. Campisi J. A novel form of the telomere-associated protein TIN2 localizes to the nuclear matrix.Cell Cycle. 2009; 8: 931-939Crossref PubMed Scopus (35) Google Scholar). Consistent with its key role in telomere maintenance, germline inactivation of TIN2 in mice is embryonic lethal (41Chiang Y.J. Kim S.H. Tessarollo L. Campisi J. Hodes R.J. Telomere-associated protein TIN2 is essential for early embryonic development through a telomerase-independent pathway.Mol. Cell. Biol. 2004; 24: 6631-6634Crossref PubMed Scopus (61) Google Scholar). Removal of TIN2 leads to the formation of telomere dysfunction-induced foci (TIFs). Importantly, clinical studies further highlight the biological significance of TIN2 in telomere protection (42Savage S.A. Giri N. Baerlocher G.M. Orr N. Lansdorp P.M. Alter B.P. TINF2, a component of the shelterin telomere protection complex, is mutated in dyskeratosis congenita.Am. J. Hum. Genet. 2008; 82: 501-509Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 43Walne A.J. Vulliamy T. Beswick R. Kirwan M. Dokal I. TINF2 mutations result in very short telomeres: Analysis of a large cohort of patients with dyskeratosis congenita and related bone marrow failure syndromes.Blood. 2008; 112: 3594-3600Crossref PubMed Scopus (227) Google Scholar). TINF2, which encodes TIN2, is the second most frequently mutated gene in the telomere elongation and protection disorder dyskeratosis congenita (DKC). DKC-associated TIN2 mutations are most frequently de novo and cluster at a highly conserved region near the end of its TRF1-binding domain. Two decades of research since the first discovery of TIN2 have shed light on protein-interaction networks around TIN2 and its multifaceted roles in telomere maintenance. 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{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{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={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{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}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.m117.776369}, abstractNote={Repair of DNA double strand breaks (DSBs) is key for maintenance of genome integrity. When DSBs are repaired by homologous recombination, DNA ends can undergo extensive processing, producing long stretches of single-stranded DNA (ssDNA). In vivo, DSB processing occurs in the context of chromatin, and studies indicate that histones may remain associated with processed DSBs. Here we demonstrate that histones are not evicted from ssDNA after in vitro chromatin resection. In addition, we reconstitute histone-ssDNA complexes (termed ssNucs) with ssDNA and recombinant histones and analyze these particles by a combination of native gel electrophoresis, sedimentation velocity, electron microscopy, and a recently developed electrostatic force microscopy technique, DREEM (dual-resonance frequency-enhanced electrostatic force microscopy). The reconstituted ssNucs are homogenous and relatively stable, and DREEM reveals ssDNA wrapping around histones. We also find that histone octamers are easily transferred in trans from ssNucs to either double-stranded DNA or ssDNA. Furthermore, the Fun30 remodeling enzyme, which has been implicated in DNA repair, binds ssNucs preferentially over nucleosomes, and ssNucs are effective at activating Fun30 ATPase activity. Our results indicate that ssNucs may be a hallmark of processes that generate ssDNA, and that posttranslational modification of ssNucs may generate novel signaling platforms involved in genome stability.}, number={13}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, 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}, month={Mar}, 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={DNA repair enzymes: structure, biophysics, and mechanism}, author={LeBlanc, S. and Wilkins, H. and Li, Z. M. and Kaur, P. and Wang, H. and Erie, D. 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}, 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}, 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}, volume={61}, ISSN={1097-2765}, url={http://dx.doi.org/10.1016/J.MOLCEL.2015.12.009}, DOI={10.1016/J.MOLCEL.2015.12.009}, abstractNote={The shelterin proteins protect telomeres against activation of the DNA damage checkpoints and recombinational repair. We show here that a dimer of the shelterin subunit TRF2 wraps ∼ 90 bp of DNA through several lysine and arginine residues localized around its homodimerization domain. The expression of a wrapping-deficient TRF2 mutant, named Top-less, alters telomeric DNA topology, decreases the number of terminal loops (t-loops), and triggers the ATM checkpoint, while still protecting telomeres against non-homologous end joining (NHEJ). In Top-less cells, the protection against NHEJ is alleviated if the expression of the TRF2-interacting protein RAP1 is reduced. We conclude that a distinctive topological state of telomeric DNA, controlled by the TRF2-dependent DNA wrapping and linked to t-loop formation, inhibits both ATM activation and NHEJ. The presence of RAP1 at telomeres appears as a backup mechanism to prevent NHEJ when topology-mediated telomere protection is impaired.}, number={2}, journal={Molecular Cell}, publisher={Elsevier BV}, 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}, month={Jan}, 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}, 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{schneeweis_obenauer-kutner_kaur_yamniuk_tamura_jaffe_o’mara_lindsay_doyle_bryson_2015, title={Comparison of Ensemble and Single Molecule Methods for Particle Characterization and Binding Analysis of a PEGylated Single-Domain Antibody}, volume={104}, ISSN={0022-3549}, url={http://dx.doi.org/10.1002/JPS.24624}, DOI={10.1002/JPS.24624}, abstractNote={Domain antibodies (dAbs) are single immunoglobulin domains that form the smallest functional unit of an antibody. This study investigates the behavior of these small proteins when covalently attached to the polyethylene glycol (PEG) moiety that is necessary for extending the half-life of a dAb. The effect of the 40 kDa PEG on hydrodynamic properties, particle behavior, and receptor binding of the dAb has been compared by both ensemble solution and surface methods [light scattering, isothermal titration calorimetry (ITC), surface Plasmon resonance (SPR)] and single-molecule atomic force microscopy (AFM) methods (topography, recognition imaging, and force microscopy). The large PEG dominates the properties of the dAb-PEG conjugate such as a hydrodynamic radius that corresponds to a globular protein over four times its size and a much reduced association rate. We have used AFM single-molecule studies to determine the mechanism of PEG-dependent reductions in the effectiveness of the dAb observed by SPR kinetic studies. Recognition imaging showed that all of the PEGylated dAb molecules are active, suggesting that some may transiently become inactive if PEG sterically blocks binding. This helps explain the disconnect between the SPR, determined kinetically, and the force microscopy and ITC results that demonstrated that PEG does not change the binding energy.}, number={12}, journal={Journal of Pharmaceutical Sciences}, publisher={Elsevier BV}, author={Schneeweis, Lumelle A. and Obenauer-Kutner, Linda and Kaur, Parminder and Yamniuk, Aaron P. and Tamura, James and Jaffe, Neil and O’Mara, Brian W. and Lindsay, Stuart and Doyle, Michael and Bryson, James}, year={2015}, month={Dec}, pages={4015–4024} }
 @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{kaur_2015, title={Enhanced Electrostatic Force Microscopy Imaging Reveals Mechanism of TRF2 Mediated DNA Compaction}, volume={108}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2014.11.914}, DOI={10.1016/J.BPJ.2014.11.914}, abstractNote={T-loop formation at telomeres is proposed to play an important role in telomere protection through the sequestration of the 3’ single-stranded overhang. Previous studies indicated that shelterin protein TRF2 modulates telomere structure by promoting DNA compaction and T-loop formation. To further understand the mechanism underlying TRF2-mediated DNA compaction, we applied Dual-Resonance-frequency-Enhanced Electrostatic force Microscopy (DREEM), a recently developed technique capable of high-resolution imaging of weak electrostatic potentials. DREEM images of nucleosomes clearly reveal DNA strands wrapped around histone proteins in nucleosomal arrays. In contrast, DREEM imaging shows DNA compacted inside TRF2 complexes through a 3-dimensional stacking of TRF2 dimers mediated by collective actions of multiple copies of TRF2 proteins. TRF2-mediated DNA compaction leads to electric potential gradients across the complexes. Surprisingly, while DNA wrapped around histones displays similar electrostatic potential signals compared to bare DNA, TRF2 DNA compaction leads to significant differences in the electrical potential signals inside multi-oligomeric TRF2 complexes compared to protein or DNA alone. These results clearly demonstrate the electrostatic changes in TRF2-DNA complexes upon oligomerizaton of proteins and DNA compaction, and underscore the importance of developing new electrostatic force microscopy imaging techniques for studying biological systems.}, number={2}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Kaur, Parminder}, year={2015}, month={Jan}, pages={166a} }
 @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{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} }
 @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{swygert_manning_senapati_kaur_lindsay_demeler_peterson_2014, title={Solution-state conformation and stoichiometry of yeast Sir3 heterochromatin fibres}, volume={5}, ISSN={2041-1723}, url={http://dx.doi.org/10.1038/NCOMMS5751}, DOI={10.1038/NCOMMS5751}, abstractNote={Heterochromatin is a repressive chromatin compartment essential for maintaining genomic integrity. A hallmark of heterochromatin is the presence of specialized nonhistone proteins that alter chromatin structure to inhibit transcription and recombination. It is generally assumed that heterochromatin is highly condensed. However, surprisingly little is known about the structure of heterochromatin or its dynamics in solution. In budding yeast, formation of heterochromatin at telomeres and the homothallic silent mating type loci require the Sir3 protein. Here, we use a combination of sedimentation velocity, atomic force microscopy and nucleosomal array capture to characterize the stoichiometry and conformation of Sir3 nucleosomal arrays. The results indicate that Sir3 interacts with nucleosomal arrays with a stoichiometry of two Sir3 monomers per nucleosome. We also find that Sir3 fibres are less compact than canonical magnesium-induced 30 nm fibres. We suggest that heterochromatin proteins promote silencing by ‘coating’ nucleosomal arrays, stabilizing interactions between nucleosomal histones and DNA. Heterochromatin is a ‘repressed’ chromatin state involved in the generation of centromeres, the protection of telomeres and the maintenance of genome integrity. Here Swygert et al.show that Sir3 - a key factor in the formation of heterochromatin - promotes a chromatin structure distinct from the canonical 30 nm fibre.}, number={1}, journal={Nature Communications}, publisher={Springer Science and Business Media LLC}, author={Swygert, Sarah G. and Manning, Benjamin J. and Senapati, Subhadip and Kaur, Parminder and Lindsay, Stuart and Demeler, Borries and Peterson, Craig L.}, year={2014}, month={Aug} }
 @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={Abstract}, 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_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}, 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{kaur_qiang-fu_fuhrmann_ros_kutner_schneeweis_navoa_steger_xie_yonan_et al._2011, title={Antibody-Unfolding and Metastable-State Binding in Force Spectroscopy and Recognition Imaging}, volume={100}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/j.bpj.2010.11.050}, DOI={10.1016/j.bpj.2010.11.050}, abstractNote={<h2>Abstract</h2> Force spectroscopy and recognition imaging are important techniques for characterizing and mapping molecular interactions. In both cases, an antibody is pulled away from its target in times that are much less than the normal residence time of the antibody on its target. The distribution of pulling lengths in force spectroscopy shows the development of additional peaks at high loading rates, indicating that part of the antibody frequently unfolds. This propensity to unfold is reversible, indicating that exposure to high loading rates induces a structural transition to a metastable state. Weakened interactions of the antibody in this metastable state could account for reduced specificity in recognition imaging where the loading rates are always high. The much weaker interaction between the partially unfolded antibody and target, while still specific (as shown by control experiments), results in unbinding on millisecond timescales, giving rise to rapid switching noise in the recognition images. At the lower loading rates used in force spectroscopy, we still find discrepancies between the binding kinetics determined by force spectroscopy and those determined by surface plasmon resonance—possibly a consequence of the short tethers used in recognition imaging. Recognition imaging is nonetheless a powerful tool for interpreting complex atomic force microscopy images, so long as specificity is calibrated in situ, and not inferred from equilibrium binding kinetics.}, number={1}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Kaur, Parminder and Qiang-Fu and Fuhrmann, Alexander and Ros, Robert and Kutner, Linda Obenauer and Schneeweis, Lumelle A. and Navoa, Ryman and Steger, Kirby and Xie, Lei and Yonan, Christopher and et al.}, year={2011}, month={Jan}, pages={243–250} }