@misc{liang_strickland_ye_wu_hu_rittschof_2019, title={Biochemistry of Barnacle Adhesion: An Updated Review}, volume={6}, ISSN={["2296-7745"]}, DOI={10.3389/fmars.2019.00565}, abstractNote={Barnacles are notorious marine fouling organisms, whose life cycle initiates with the planktonic larva, followed by the free-swimming cyprid that voluntarily explores and searches for an appropriate site to settle and metamorphoses into a sessile adult. Within this life cycle, both the cyprid and the adult barnacle deposit multi-protein adhesives for temporary or permanent underwater adhesion. Here, we present a comprehensive review of the biochemistries behind these different adhesion events in the life cycle of a barnacle. First, we introduce the multiple adhesion events and their corresponding adhesives from two complementary aspects: the in vivo synthesis, storage, and secretion as well as the in vitro morphology and biochemistry. The amino acid compositions, sequences, and structures of adult barnacle adhesive proteins are specifically highlighted. Second, we discuss the molecular mechanisms of adult barnacle underwater attachment in detail by analyzing the possible adhesive and cohesive roles of different adhesive proteins, and based on these analyses, we propose an update to the original barnacle underwater adhesion molecular model. We believe that this review can greatly promote the general understanding of the molecular mechanisms underlying the reversible and irreversible underwater adhesion of barnacles and their larvae. Such an understanding is the basis for the prevention of barnacle fouling on target surfaces as well as designing conceptually new barnacle-inspired artificial underwater adhesives.}, journal={FRONTIERS IN MARINE SCIENCE}, author={Liang, Chao and Strickland, Jack and Ye, Zonghuang and Wu, Wenjian and Hu, Biru and Rittschof, Dan}, year={2019}, month={Sep} }
 @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. 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. In eukaryotes, proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids (1Michaelis C. Ciosk R. Nasmyth K. Cohesins: chromosomal proteins that prevent premature separation of sister chromatids.Cell. 1997; 91 (9335333): 35-4510.1016/S0092-8674(01)80007-6Abstract Full Text Full Text PDF PubMed Scopus (1168) Google Scholar, 2Uhlmann F. Nasmyth K. Cohesion between sister chromatids must be established during DNA replication.Curr. Biol. 1998; 8 (9778527): 1095-110110.1016/S0960-9822(98)70463-4Abstract Full Text Full Text PDF PubMed Google Scholar). Cohesion is mediated by the cohesin complex, which also plays important roles in diverse biological processes, including DNA double-strand break (DSB) 2The abbreviations used are: DSBdouble-strand breakAFMatomic force microscopyQDquantum dotHRhomologous recombinationntnucleotideNTAnitrilotriacetic acidBTtris-NTAbiotinylated tris-nitrilotriacetic acidMSDmean square displacement. repair, restart of stalled replication forks, and maintenance of 3D chromatin organization (3Bose T. Gerton J.L. Cohesinopathies, gene expression, and chromatin organization.J. Cell Biol. 2010; 189 (20404106): 201-21010.1083/jcb.200912129Crossref PubMed Scopus (76) Google Scholar, 4Nasmyth K. Haering C.H. Cohesin: its roles and mechanisms.Annu. Rev. Genet. 2009; 43 (19886810): 525-55810.1146/annurev-genet-102108-134233Crossref PubMed Scopus (722) Google Scholar). In vertebrates, cohesin consists of heterodimeric ATPases SMC1 and SMC3, a kleisin subunit RAD21 (also known as Scc1), and the stromal antigen (SA or Heat-B) subunit, which can be either SA1 (STAG1) or SA2 (STAG2). The core cohesin complex exists at 1:1:1:1 stoichiometry in cells (5Holzmann J. Fuchs J. Pichler P. Peters J.M. Mechtler K. Lesson from the stoichiometry determination of the cohesin complex: a short protease mediated elution increases the recovery from cross-linked antibody-conjugated beads.J. Proteome Res. 2011; 10 (21043528): 780-78910.1021/pr100927xCrossref PubMed Scopus (18) Google Scholar). Electron microscopy–, crystallography–, and biochemical assay–based studies support the notion that cohesin binds to DNA by topological embrace through the ring subunits (SMC1, SMC3, and RAD21) (6Gruber S. Haering C.H. Nasmyth K. Chromosomal cohesin forms a ring.Cell. 2003; 112 (12654244): 765-77710.1016/S0092-8674(03)00162-4Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar7Haering C.H. Löwe J. Hochwagen A. Nasmyth K. Molecular architecture of SMC proteins and the yeast cohesin complex.Mol. Cell. 2002; 9 (11983169): 773-78810.1016/S1097-2765(02)00515-4Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar, 8Ivanov D. Nasmyth K. A topological interaction between cohesin rings and a circular minichromosome.Cell. 2005; 122 (16179255): 849-86010.1016/j.cell.2005.07.018Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 9Murayama Y. Uhlmann F. Biochemical reconstitution of topological DNA binding by the cohesin ring.Nature. 2014; 505 (24291789): 367-371Crossref PubMed Scopus (195) Google Scholar, 10Skibbens R.V. Cell biology: cohesin rings leave loose ends.Curr. Biol. 2015; 25 (25649818): R108-R11010.1016/j.cub.2014.12.015Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar11Skibbens R.V. Of rings and rods: regulating cohesin entrapment of DNA to generate intra- and intermolecular tethers.PLoS Genet. 2016; 12 (27788133): e100633710.1371/journal.pgen.1006337Crossref PubMed Scopus (23) Google Scholar). SA1 and SA2 share 70% sequence homology and exist in separate cohesin complexes, with SA2 being more abundant than SA1 (12Carramolino L. Lee B.C. Zaballos A. Peled A. Barthelemy I. Shav-Tal Y. Prieto I. Carmi P. Gothelf Y. González de Buitrago G. Aracil M. Márquez G. Barbero J.L. Zipori D. SA-1, a nuclear protein encoded by one member of a novel gene family: molecular cloning and detection in hemopoietic organs.Gene. 1997; 195 (9305759): 151-15910.1016/S0378-1119(97)00121-2Crossref PubMed Scopus (36) Google Scholar, 13Losada A. Yokochi T. Kobayashi R. Hirano T. Identification and characterization of SA/Scc3p subunits in the Xenopus and human cohesin complexes.J. Cell Biol. 2000; 150 (10931856): 405-41610.1083/jcb.150.3.405Crossref PubMed Scopus (258) Google Scholar14Sumara I. Vorlaufer E. Gieffers C. Peters B.H. Peters J.M. Characterization of vertebrate cohesin complexes and their regulation in prophase.J. Cell Biol. 2000; 151 (11076961): 749-76210.1083/jcb.151.4.749Crossref PubMed Scopus (336) Google Scholar). In addition to the core cohesin subunits, several cohesin regulatory factors have been discovered that play important roles in the loading, stability, and cleavage of the cohesin ring during different phases of the cell cycle (15Rollins R.A. Korom M. Aulner N. Martens A. Dorsett D. Drosophila nipped-B protein supports sister chromatid cohesion and opposes the stromalin/Scc3 cohesion factor to facilitate long-range activation of the cut gene.Mol. Cell. Biol. 2004; 24 (15060134): 3100-311110.1128/MCB.24.8.3100-3111.2004Crossref PubMed Scopus (187) Google Scholar16Tedeschi A. Wutz G. Huet S. Jaritz M. Wuensche A. Schirghuber E. Davidson I.F. Tang W. Cisneros D.A. Bhaskara V. Nishiyama T. Vaziri A. Wutz A. Ellenberg J. Peters J.M. Wapl is an essential regulator of chromatin structure and chromosome segregation.Nature. 2013; 501 (23975099): 564-56810.1038/nature12471Crossref PubMed Scopus (196) Google Scholar, 17Carretero M. Ruiz-Torres M. Rodríguez-Corsino M. Barthelemy I. Losada A. Pds5B is required for cohesion establishment and Aurora B accumulation at centromeres.EMBO J. 2013; 32 (24141881): 2938-294910.1038/emboj.2013.230Crossref PubMed Scopus (65) Google Scholar18Zhang N. Kuznetsov S.G. Sharan S.K. Li K. Rao P.H. Pati D. A handcuff model for the cohesin complex.J. Cell Biol. 2008; 183 (19075111): 1019-103110.1083/jcb.200801157Crossref PubMed Scopus (124) Google Scholar). Furthermore, non-SMC subunits in cohesin and condensin (Psc3, Ycg1, and Ycs4) and NSE1/3/4 from the SMC5/6 complex have been implicated in DNA binding (9Murayama Y. Uhlmann F. Biochemical reconstitution of topological DNA binding by the cohesin ring.Nature. 2014; 505 (24291789): 367-371Crossref PubMed Scopus (195) Google Scholar, 19Piazza I. Rutkowska A. Ori A. Walczak M. Metz J. Pelechano V. Beck M. Haering C.H. Association of condensin with chromosomes depends on DNA binding by its HEAT-repeat subunits.Nat. Struct. Mol. Biol. 2014; 21 (24837193): 560-56810.1038/nsmb.2831Crossref PubMed Scopus (76) Google Scholar, 20Zabrady K. Adamus M. Vondrova L. Liao C. Skoupilova H. Novakova M. Jurcisinova L. Alt A. Oliver A.W. Lehmann A.R. Palecek J.J. Chromatin association of the SMC5/6 complex is dependent on binding of its NSE3 subunit to DNA.Nucleic Acids Res. 2016; 44 (26446992): 1064-107910.1093/nar/gkv1021Crossref PubMed Scopus (47) Google Scholar). double-strand break atomic force microscopy quantum dot homologous recombination nucleotide nitrilotriacetic acid biotinylated tris-nitrilotriacetic acid mean square displacement. Germ line mutations in core cohesin subunits or their regulators are associated with a spectrum of human diseases collectively called “cohesinopathies” and an increased incidence of cancer (3Bose T. Gerton J.L. Cohesinopathies, gene expression, and chromatin organization.J. Cell Biol. 2010; 189 (20404106): 201-21010.1083/jcb.200912129Crossref PubMed Scopus (76) Google Scholar, 21Solomon D.A. Kim J.S. Waldman T. Cohesin gene mutations in tumorigenesis: from discovery to clinical significance.BMB Rep. 2014; 47 (24856830): 299-31010.5483/BMBRep.2014.47.6.092Crossref PubMed Scopus (47) Google Scholar, 22Watrin E. Kaiser F.J. Wendt K.S. Gene regulation and chromatin organization: relevance of cohesin mutations to human disease.Curr. Opin. Genet. Dev. 2016; 37 (26821365): 59-6610.1016/j.gde.2015.12.004Crossref PubMed Scopus (49) Google Scholar). Somatic mutations of the SA2 gene and loss of SA2 protein expression have been reported in multiple cancer cell lines, including urothelial bladder carcinomas, Ewing’s sarcomas, glioblastomas, and malignant melanomas (21Solomon D.A. Kim J.S. Waldman T. Cohesin gene mutations in tumorigenesis: from discovery to clinical significance.BMB Rep. 2014; 47 (24856830): 299-31010.5483/BMBRep.2014.47.6.092Crossref PubMed Scopus (47) Google Scholar). Despite the progress made since the discovery of the cohesin complex, many fundamental questions regarding the structure and assembly of cohesin remain unanswered (23Onn I. Heidinger-Pauli J.M. Guacci V. Unal E. Koshland D.E. Sister chromatid cohesion: a simple concept with a complex reality.Annu. Rev. Cell Dev. Biol. 2008; 24 (18616427): 105-12910.1146/annurev.cellbio.24.110707.175350Crossref PubMed Scopus (249) Google Scholar, 24Skibbens R.V. Buck the establishment: reinventing sister chromatid cohesion.Trends Cell Biol. 2010; 20 (20620062): 507-51310.1016/j.tcb.2010.06.003Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). For example, how cohesin binds to chromatin to establish sister chromatid cohesion is not fully understood (25Uhlmann F. A matter of choice: the establishment of sister chromatid cohesion.EMBO Rep. 2009; 10 (19745840): 1095-110210.1038/embor.2009.207Crossref PubMed Scopus (34) Google Scholar). Various models, including one ring, twin-ring handcuffs, bracelet oligomers, and C-clamps, have been proposed for cohesin assembly on DNA (24Skibbens R.V. Buck the establishment: reinventing sister chromatid cohesion.Trends Cell Biol. 2010; 20 (20620062): 507-51310.1016/j.tcb.2010.06.003Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). However, these models have not taken into consideration that SA2 plays important roles both in stabilizing cohesin on DNA and unloading cohesin from chromatin. It is known that SA2 phosphorylation by the polo-like kinase 1 (Plk1) leads to the removal of cohesin from chromatin (26Hauf S. Roitinger E. Koch B. Dittrich C.M. Mechtler K. Peters J.M. Dissociation of cohesin from chromosome arms and loss of arm cohesion during early mitosis depends on phosphorylation of SA2.PLoS Biol. 2005; 3 (15737063): e6910.1371/journal.pbio.0030069Crossref PubMed Scopus (362) Google Scholar), indicating the importance of SA2 in the relationship of cohesin with DNA. In addition, how cohesin DNA binding is spatially controlled along the genome is poorly understood. DNA DSB induction leads to the establishment of sister chromatid cohesion in the G2 phase, which facilitates the DNA repair process (27Ström L. Lindroos H.B. Shirahige K. Sjögren C. Postreplicative recruitment of cohesin to double-strand breaks is required for DNA repair.Mol. Cell. 2004; 16 (15610742): 1003-101510.1016/j.molcel.2004.11.026Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar28Ström L. Karlsson C. Lindroos H.B. Wedahl S. Katou Y. Shirahige K. Sjögren C. Postreplicative formation of cohesion is required for repair and induced by a single DNA break.Science. 2007; 317 (17626884): 242-24510.1126/science.1140649Crossref PubMed Scopus (236) Google Scholar, 29Bauerschmidt C. Arrichiello C. Burdak-Rothkamm S. Woodcock M. Hill M.A. Stevens D.L. Rothkamm K. Cohesin promotes the repair of ionizing radiation-induced DNA double-strand breaks in replicated chromatin.Nucleic Acids Res. 2010; 38 (19906707): 477-48710.1093/nar/gkp976Crossref PubMed Scopus (72) Google Scholar, 30Cortés-Ledesma F. Aguilera A. Double-strand breaks arising by replication through a nick are repaired by cohesin-dependent sister-chromatid exchange.EMBO Rep. 2006; 7 (16888651): 919-92610.1038/sj.embor.7400774Crossref PubMed Scopus (119) Google Scholar31Sjögren C. Nasmyth K. Sister chromatid cohesion is required for postreplicative double-strand break repair in Saccharomyces cerevisiae.Curr. Biol. 2001; 11 (11448778): 991-99510.1016/S0960-9822(01)00271-8Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). It was proposed that following the induction of DSBs, cohesin is recruited to the region surrounding the DSB as well as genome-wide through the DNA damage response pathway and chromatin remodeling (32Unal E. Arbel-Eden A. Sattler U. Shroff R. Lichten M. Haber J.E. Koshland D. DNA damage response pathway uses histone modification to assemble a double-strand break-specific cohesin domain.Mol. Cell. 2004; 16 (15610741): 991-100210.1016/j.molcel.2004.11.027Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 33Unal E. Heidinger-Pauli J.M. Koshland D. DNA double-strand breaks trigger genome-wide sister-chromatid cohesion through Eco1 (Ctf7).Science. 2007; 317 (17626885): 245-24810.1126/science.1140637Crossref PubMed Scopus (252) Google Scholar). In addition, the Schizosaccharomyces pombe cohesin ring is capable of sliding on DNA with a diffusion constant approaching the theoretical limit for free 1D diffusion, and the complex falls off from free DNA ends (34Stigler J. Çamdere G.Ö. Koshland D.E. Greene E.C. Single-molecule imaging reveals a collapsed conformational state for DNA-bound cohesin.Cell Rep. 2016; 15 (27117417): 988-99810.1016/j.celrep.2016.04.003Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). These observations raise an important question: How does the cohesin complex promote stable cohesion during DNA DSB repair without sliding off from DNA ends? In addition, SA1 and SA2 have different roles during DSB repair, as well as during sister chromatid cohesion at telomeres and centromeres (35Canudas 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 (17962804): 4867-487810.1038/sj.emboj.7601903Crossref PubMed Scopus (82) Google Scholar, 36Bisht K.K. Daniloski Z. Smith S. SA1 binds directly to DNA through its unique AT-hook to promote sister chromatid cohesion at telomeres.J. Cell Sci. 2013; 126 (23729739): 3493-350310.1242/jcs.130872Crossref PubMed Scopus (28) Google Scholar). Whereas SA2 is important for cohesion at centromeres, depletion analysis showed that telomeres relied heavily on SA1 and to a lesser extent on the cohesin ring for cohesion (35Canudas 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 (17962804): 4867-487810.1038/sj.emboj.7601903Crossref PubMed Scopus (82) Google Scholar, 36Bisht K.K. Daniloski Z. Smith S. SA1 binds directly to DNA through its unique AT-hook to promote sister chromatid cohesion at telomeres.J. Cell Sci. 2013; 126 (23729739): 3493-350310.1242/jcs.130872Crossref PubMed Scopus (28) Google Scholar). It has been suggested that the SA subunits in humans and their orthologs in yeast (Scc3 in budding yeast and Psc3 in fission yeast) play a role in the loading of cohesin ring onto chromosomes through the interaction between DNA and the cohesin hinge (37Huis in 't Veld P.J. Herzog F. Ladurner R. Davidson I.F. Piric S. Kreidl E. Bhaskara V. Aebersold R. Peters J.M. Characterization of a DNA exit gate in the human cohesin ring.Science. 2014; 346 (25414306): 968-97210.1126/science.1256904Crossref PubMed Scopus (128) Google Scholar, 38Murayama Y. Uhlmann F. DNA entry into and exit out of the cohesin ring by an interlocking gate mechanism.Cell. 2015; 163 (26687354): 1628-164010.1016/j.cell.2015.11.030Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The crystal structure of SA2 (residues 80–1060) shows that it contains a helical domain at its N terminus followed by 17 HEAT repeats shaped like a dragon (39Hara K. Zheng G. Qu Q. Liu H. Ouyang Z. Chen Z. Tomchick D.R. Yu H. Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion.Nat. Struct. Mol. Biol. 2014; 21 (25173175): 864-87010.1038/nsmb.2880Crossref PubMed Scopus (101) Google Scholar, 40Roig M.B. Löwe J. Chan K.L. Beckouët F. Metson J. Nasmyth K. Structure and function of cohesin's Scc3/SA regulatory subunit.FEBS Lett. 2014; 588 (25171859): 3692-370210.1016/j.febslet.2014.08.015Crossref PubMed Scopus (48) Google Scholar). Binding to DNA through the HEAT repeat–containing subunits has been proposed to serve as the first step in condensin loading (19Piazza I. Rutkowska A. Ori A. Walczak M. Metz J. Pelechano V. Beck M. Haering C.H. Association of condensin with chromosomes depends on DNA binding by its HEAT-repeat subunits.Nat. Struct. Mol. Biol. 2014; 21 (24837193): 560-56810.1038/nsmb.2831Crossref PubMed Scopus (76) Google Scholar). The N- and C-terminal domains of SA1 and SA2 share only 30–50% homology, which makes it likely that these domains contribute to their functional specificities. Recently, we discovered that SA1 binds to dsDNA and shows specificity for telomeric sequences (41Lin 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 (27298259): 6363-637610.1093/nar/gkw518Crossref PubMed Scopus (21) Google Scholar). These new results raise an important question as to whether or not SA2 specifically recognizes unique DNA sequences or structures. Here, to investigate the binding of SA2 to specific DNA sequences and structures, we applied fluorescence anisotropy and two complementary single-molecule imaging techniques, atomic force microscopy (AFM) and fluorescence imaging of quantum dot (QD)-labeled proteins on DNA tightropes. In contrast to SA1 (41Lin 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 (27298259): 6363-637610.1093/nar/gkw518Crossref PubMed Scopus (21) Google Scholar), the 1D diffusion dynamics of SA2 on DNA is independent of telomeric or centromeric sequences. Fluorescence anisotropy shows that SA2 binds to both ssDNA and dsDNA, albeit with a higher binding affinity for ssDNA. In addition, SA2 recognizes DNA overhang, flap, and fork, which are intermediate DNA structures during DNA repair, recombination, and replication. Likewise, AFM imaging reveals that SA2 displays high binding specificities for the DNA end, ssDNA gap, flap, single-stranded fork, and replication fork. Strikingly, SA2 is capable of switching between two DNA-binding modes: searching through unbiased 1D diffusion on dsDNA and recognition through stable binding at the ssDNA gap. Furthermore, results from the DR-GFP reporter system show that SA2 directly facilitates homologous recombination (HR)-mediated DNA DSB repair. Importantly, these results strongly suggest a new role for SA2 in recognizing intermediate DNA structures during genome maintenance pathways. Studying the DNA-binding properties of SA1 and SA2 is essential for advancing our understanding of the function of the cohesin complex in diverse genome maintenance pathways. Recently, we discovered that SA1 binds to DNA through the AT-hook domain at its N-terminal domain (41Lin 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 (27298259): 6363-637610.1093/nar/gkw518Crossref PubMed Scopus (21) Google Scholar). SA2 lacks the AT-hook motif (36Bisht K.K. Daniloski Z. Smith S. SA1 binds directly to DNA through its unique AT-hook to promote sister chromatid cohesion at telomeres.J. Cell Sci. 2013; 126 (23729739): 3493-350310.1242/jcs.130872Crossref PubMed Scopus (28) Google Scholar). To investigate whether or not SA2 is a DNA-binding protein, we purified His-tagged full-length SA2 (Fig. 1A and Methods). First, we evaluated the oligomeric state of SA2 using a previously established method that estimates the molecular mass of a protein based on the calibration curve correlating AFM volume and molecular weight of proteins (42Yang Y. Wang H. Erie D.A. Quantitative characterization of biomolecular assemblies and interactions using atomic force microscopy.Methods. 2003; 29 (12606223): 175-18710.1016/S1046-2023(02)00308-0Crossref PubMed Scopus (89) Google Scholar, 43Fuentes-Perez M.E. Dillingham M.S. Moreno-Herrero F. AFM volumetric methods for the characterization of proteins and nucleic acids.Methods. 2013; 60 (23454289): 113-12110.1016/j.ymeth.2013.02.005Crossref PubMed Scopus (32) Google Scholar44Kaur 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 (26856421): 2051310.1038/srep20513Crossref PubMed Scopus (22) Google Scholar). Based on this method, SA2 molecules (141 kDa) display AFM volumes (146 nm3) consistent with being predominantly monomers (Fig. S1A). This result is consistent with our earlier analysis of SA2 molecular weight using gel filtration chromatography (45Zhang N. Jiang Y. Mao Q. Demeler B. Tao Y.J. Pati D. Characterization of the interaction between the cohesin subunits Rad21 and SA1/2.PLoS One. 2013; 8 (23874961): e6945810.1371/journal.pone.0069458Crossref PubMed Scopus (26) Google Scholar). To evaluate SA2-DNA binding specificity, we applied AFM imaging of SA2 in the presence of linear DNA fragments containing either centromeric or telomeric sequences (Fig. 1A and Methods). Ensemble-based biochemical assays, such as fluorescence anisotropy and EMSAs, only provide average binding affinities for DNA substrates. These assays cannot differentiate sequence-specific DNA binding from DNA end binding. In contrast, from AFM images of protein-DNA complexes, a direct measurement of the DNA-binding specificity for unique sequences as well as that for DNA structures such as ends can be obtained through statistical analysis of binding positions of protein complexes on individual DNA fragments (46Yang Y. Sass L.E. Du C. Hsieh P. Erie D.A. Determination of protein-DNA binding constants and specificities from statistical analyses of single molecules: MutS-DNA interactions.Nucleic Acids Res. 2005; 33 (16061937): 4322-433410.1093/nar/gki708Crossref PubMed Scopus (85) Google Scholar). Two centromeric DNA substrates (4.1 kb) used for AFM imaging contain the α-satellite centromeric sequences that are either close to one end of the linearized DNA (Cen-end DNA) or near the middle (Cen-mid DNA) (Fig. 1A). For the telomeric DNA substrate (T270 DNA), the (TTAGGG)270 sequences make up ∼30% of the total DNA length (5.4 kb) and are located at the middle of the linearized T270 DNA (Fig. 1A). SA2 molecules displayed AFM heights (1.41 ± 0.30 nm, mean ± S.D., Fig. 1 (B and C) and supplemental Fig. S1B) that were significantly taller than that of dsDNA alone (0.70 ± 0.08 nm, mean ± S.D.). This large difference in heights enabled unambiguous identification of SA2 molecules on DNA. Statistical analysis of the binding position of SA2 on DNA revealed that SA2 did not bind specifically to either the centromeric or telomeric sequences (Fig. 1D). However, on all three DNA substrates, the majority of SA2 molecules were bound at the DNA ends. Furthermore, DNA end binding by SA2 was independent of the internal DNA sequence, the position of the centromeric region, or the presence of single-stranded overhangs at the terminal ends (4-nt 3′-overhang on Cen-end DNA; Fig. 1D). To further quantify the SA2-binding specificity for DNA ends, we applied the analysis based on the fractional occupancies of SA2 at DNA ends (46Yang Y. Sass L.E. Du C. Hsieh P. Erie D.A. Determination of protein-DNA binding constants and specificities from statistical analyses of single molecules: MutS-DNA interactions.Nucleic Acids Res. 2005; 33 (16061937): 4322-433410.1093/nar/gki708Crossref PubMed Scopus (85) Google Scholar). SA2-binding specificities for DNA ends (S = DNA binding constant for specific sites/DNA binding constant for nonspecific sites = KSP/KNSP) are 2945 ± 77, 2604 ± 68, and 2129 ± 76, respectively, for T270, Cen-end, and Cen-mid DNA substrates. In addition, in contrast to SA2 alone, DNA-bound SA2 formed higher-order oligomeric complexes with average AFM volumes of 1025 ± 88 and 898 ± 63 nm3, respectively, at DNA ends and internal sites (Fig. S1C). Based on the calibration curve relating protein molecular weights and AFM volumes (44Kaur 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 (26856421): 2051310.1038/srep20513Crossref PubMed Scopus (22) Google Scholar), these AFM volumes correspond to approximately five and four SA2 molecules, respectively, at the DNA ends and internal sites. In summary, SA2 does not specifically bind to centromeric sequences, but binds DNA ends with high specificities that are independent of DNA sequences and short (4-nt) single-stranded overhangs. Previously, it was established that cohesin deposition and establishment occur in concert with lagging strand-processing (47Rudra S. Skibbens R.V. Sister chromatid cohesion establishment occurs in concert with lagging strand synthesis.Cell Cycle. 2012; 11 (22592531): 2114-212110.4161/cc.20547Crossref PubMed Scopus (38) Google Scholar). ssDNA gaps are intermediate structures on lagging strand during DNA replication. To directly test whether or not SA2 binds to ssDNA gaps, we used a previously established method to generate a linear substrate containing an ssDNA gap (37 nt) flanked by dsDNA arms (Fig. 2A). This method was based on the generation of four closely spaced nicks using DNA nickase and subsequent removal of short ssDNA between nicked sites using complementary oligonucleotides (48Geng H. Du C. Chen S. Salerno V. Manfredi C. Hsieh P. In vitro studies of DNA mismatch repair proteins.Anal. Biochem. 2011; 413 (21329650): 179-18410.1016/j.ab.2011.02.017Crossref PubMed Scopus (25) Google Scholar, 49Buechner C.N. Tessmer I. DNA substrate preparation for atomic force microscopy studies of protein-DNA interactions.J. Mol. Recognit. 2013; 26 (24277605): 605-61710.1002/jmr.2311Crossref PubMed Scopus (27) Google Scholar). After restriction digestion of the circular gapped DNA, the ssDNA gap is at 470 nt (23%) from one end of the DNA (blunt end; Fig. 2A and Fig. S2A). Based on diagnostic restriction digestion at the gapped region, DNA gapping efficiencies were typically 85–95% (Fig. S2B). To further confirm the presence of the ssDNA gap, the position distribution of mitochondrial single-stranded DNA-binding protein on this DNA substrate was analyzed. Mitochondrial single-stranded DNA-binding protein predominantly bound to the expected ssDNA region on the gapped DNA substrate, whereas its binding on the nicked DNA substrate was random. 3P. Kaur and H. Wang, unpublished data. In summary, these results est}, 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} }