2023 journal article

High-speed AFM imaging reveals DNA capture and loop extrusion dynamics by cohesin-NIPBL

JOURNAL OF BIOLOGICAL CHEMISTRY, 299(11).

By: P. Kaur n, X. Lu *, Q. Xu *, E. Irvin n, C. Pappas n, H. Zhang *, I. Finkelstein *, Z. Shi * ...

TL;DR: 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). (via Semantic Scholar)
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Added: December 11, 2023

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. 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. Large-scale spatial segregation of open and closed chromatin compartments and topologically associating domains (TADs), sub-TADs, and loops fold the genome in interphase (1Lieberman-Aiden E. van Berkum N.L. Williams L. Imakaev M. Ragoczy T. Telling A. et al.Comprehensive mapping of long-range interactions reveals folding principles of the human genome.Science. 2009; 326: 289-293Crossref PubMed Scopus (5428) Google Scholar, 2Dixon J.R. Selvaraj S. Yue F. Kim A. Li Y. Shen Y. et al.Topological domains in mammalian genomes identified by analysis of chromatin interactions.Nature. 2012; 485: 376-380Crossref PubMed Scopus (4342) Google Scholar, 3Nora E.P. Lajoie B.R. Schulz E.G. Giorgetti L. Okamoto I. 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Genet. 2019; 53: 445-482Crossref PubMed Scopus (153) Google Scholar). The core cohesin complex includes SMC1, SMC3, RAD21Scc1, and SA1/SA2Scc3 (humanyeast, Fig. 1A). SMC proteins (SMC1 and SMC3) form long antiparallel coiled coils (arms), each with a dimerization (hinge) domain at one end and an ABC-type ATPase (head) domain at the other. RAD21Scc1 interconnects the head domains. In addition, SA1 and SA2 (STAG1 and STAG2) directly interact with the CCCTC-binding factor (CTCF), a ubiquitous zinc-finger protein that specifically localizes to CTCF-binding sites along the genome (14Xiao T. Wallace J. Felsenfeld G. Specific sites in the C terminus of CTCF interact with the SA2 subunit of the cohesin complex and are required for cohesin-dependent insulation activity.Mol. Cell. Biol. 2011; 31: 2174-2183Crossref PubMed Scopus (144) Google Scholar). Though initially identified as an essential complex to hold sister chromatids together (15Nasmyth K. Haering C.H. Cohesin: its roles and mechanisms.Annu. Rev. Genet. 2009; 43: 525-558Crossref PubMed Scopus (754) Google Scholar), numerous studies demonstrated that cohesin is also crucial in mediating 3D chromatin organization during interphase (16Sofueva S. Yaffe E. Chan W.C. Georgopoulou D. Vietri Rudan M. Mira-Bontenbal H. et al.Cohesin-mediated interactions organize chromosomal domain architecture.EMBO J. 2013; 32: 3119-3129Crossref PubMed Scopus (298) Google Scholar, 17Guo Y. Xu Q. Canzio D. Shou J. Li J. Gorkin D.U. et al.CRISPR inversion of CTCF sites alters genome topology and enhancer/promoter function.Cell. 2015; 162: 900-910Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar, 18Narendra V. Rocha P.P. An D. Raviram R. Skok J.A. Mazzoni E.O. et al.CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation.Science. 2015; 347: 1017-1021Crossref PubMed Scopus (382) Google Scholar, 19Phillips-Cremins J.E. Sauria M.E. Sanyal A. Gerasimova T.I. Lajoie B.R. Bell J.S. et al.Architectural protein subclasses shape 3D organization of genomes during lineage commitment.Cell. 2013; 153: 1281-1295Abstract Full Text Full Text PDF PubMed Scopus (859) Google Scholar, 20Seitan V.C. Banks P. Laval S. Majid N.A. Dorsett D. Rana A. et al.Metazoan Scc4 homologs link sister chromatid cohesion to cell and axon migration guidance.PLoS Biol. 2006; 4: e242Crossref PubMed Scopus (87) Google Scholar, 21Zuin J. Dixon J.R. van der Reijden M.I. Ye Z. Kolovos P. Brouwer R.W. et al.Cohesin and CTCF differentially affect chromatin architecture and gene expression in human cells.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 996-1001Crossref PubMed Scopus (565) Google Scholar). Greater than 80% of long-range looping interactions are mediated by some combinations of cohesin, CTCF, and the mediator complex. Cohesin and CTCF are enriched at TAD boundaries and corner peaks that indicate strong interactions at TAD borders (2Dixon J.R. Selvaraj S. Yue F. Kim A. Li Y. Shen Y. et al.Topological domains in mammalian genomes identified by analysis of chromatin interactions.Nature. 2012; 485: 376-380Crossref PubMed Scopus (4342) Google Scholar, 5Rao S.S. Huntley M.H. Durand N.C. Stamenova E.K. Bochkov I.D. Robinson J.T. et al.A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping.Cell. 2014; 159: 1665-1680Abstract Full Text Full Text PDF PubMed Scopus (4247) Google Scholar). Furthermore, NIPBL significantly stimulates cohesin's DNA binding and ATPase activities (22Kim Y. Shi Z. Zhang H. Finkelstein I.J. Yu H. Human cohesin compacts DNA by loop extrusion.Science. 2019; 366: 1345-1349Crossref PubMed Scopus (347) Google Scholar). RAD21 or NIPBL depletion leads to significantly reduced TADs and corner peaks. A large body of literature supports a model that cohesin-NIPBL mediates TAD and chromatin loop formation through DNA loop extrusion (23Davidson I.F. Bauer B. Goetz D. Tang W. Wutz G. Peters J.M. DNA loop extrusion by human cohesin.Science. 2019; 366: 1338-1345Crossref PubMed Scopus (391) Google Scholar, 24Banigan E.J. Mirny L.A. The interplay between asymmetric and symmetric DNA loop extrusion.Elife. 2020; 9: e63528Crossref PubMed Scopus (11) Google Scholar, 25Bauer B.W. Davidson I.F. Canena D. Wutz G. Tang W. Litos G. et al.Cohesin mediates DNA loop extrusion by a "swing and clamp" mechanism.Cell. 2021; 184: 5448-5464.e5422Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 26Higashi T.L. Pobegalov G. Tang M. Molodtsov M.I. Uhlmann F. A Brownian ratchet model for DNA loop extrusion by the cohesin complex.Elife. 2021; 10: e67530Crossref PubMed Scopus (35) Google Scholar). The DNA loop extrusion model posits that cohesin creates DNA loops by actively extruding DNA until they are stabilized by CTCF bound at converging CTCF-binding sites (27Fudenberg G. Abdennur N. Imakaev M. Goloborodko A. Mirny L.A. Emerging evidence of chromosome folding by loop extrusion.Cold Spring Harb. Symposia Quant. Biol. 2017; 82: 45-55Crossref PubMed Scopus (147) Google Scholar, 28Fudenberg G. Imakaev M. Lu C. Goloborodko A. Abdennur N. Mirny L.A. Formation of chromosomal domains by loop extrusion.Cell Rep. 2016; 15: 2038-2049Abstract Full Text Full Text PDF PubMed Scopus (1061) Google Scholar). Importantly, single-molecule fluorescence imaging studies, including ours, demonstrated that cohesin-NIPBL is capable of DNA loop extrusion in an ATPase-dependent manner (22Kim Y. Shi Z. Zhang H. Finkelstein I.J. Yu H. Human cohesin compacts DNA by loop extrusion.Science. 2019; 366: 1345-1349Crossref PubMed Scopus (347) Google Scholar, 23Davidson I.F. Bauer B. Goetz D. Tang W. Wutz G. Peters J.M. DNA loop extrusion by human cohesin.Science. 2019; 366: 1338-1345Crossref PubMed Scopus (391) Google Scholar). Several unique features of the cohesin-NIPBL structure have implications in its mechanism of action. Cohesin-NIPBL contains DNA-binding sites on multiple subunits with DNA-binding affinities that differ by two orders of magnitudes (25Bauer B.W. Davidson I.F. Canena D. Wutz G. Tang W. Litos G. et al.Cohesin mediates DNA loop extrusion by a "swing and clamp" mechanism.Cell. 2021; 184: 5448-5464.e5422Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Previous high-speed atomic force microscopy (HS-AFM) imaging also showed that cohesin and condensin are capable of significant conformational changes. These include SMC ring opening and closing, alignment of the SMC arms, elbow bending, and SMC head engagement and disengagement (25Bauer B.W. Davidson I.F. Canena D. Wutz G. Tang W. Litos G. et al.Cohesin mediates DNA loop extrusion by a "swing and clamp" mechanism.Cell. 2021; 184: 5448-5464.e5422Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 29Anderson D.E. Losada A. Erickson H.P. Hirano T. Condensin and cohesin display different arm conformations with characteristic hinge angles.J. Cell Biol. 2002; 156: 419-424Crossref PubMed Scopus (305) Google Scholar, 30Eeftens J.M. Katan A.J. Kschonsak M. Hassler M. de Wilde L. Dief E.M. et al.Condensin Smc2-Smc4 dimers are flexible and dynamic.Cell Rep. 2016; 14: 1813-1818Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 31Sedeno Cacciatore A. Rowland B.D. Loop formation by SMC complexes: turning heads, bending elbows, and fixed anchors.Curr. Opin. Genet. Dev. 2019; 55: 11-18Crossref PubMed Scopus (19) Google Scholar, 32Burmann F. Lee B.G. Than T. Sinn L. O'Reilly F.J. Yatskevich S. et al.A folded conformation of MukBEF and cohesin.Nat. Struct. Mol. Biol. 2019; 26: 227-236Crossref PubMed Scopus (75) Google Scholar, 33Ryu J.K. Katan A.J. van der Sluis E.O. Wisse T. de Groot R. Haering C.H. et al.The condensin holocomplex cycles dynamically between open and collapsed states.Nat. Struct. Mol. Biol. 2020; 27: 1134-1141Crossref PubMed Scopus (40) Google Scholar). To achieve DNA loop extrusion, cohesin-NIPBL in solution needs first to capture DNA, followed by anchoring onto DNA while still capable of reeling in DNA to enlarge the DNA loop. Observations from single-molecule fluorescence imaging did not provide information on protein conformational changes that drive DNA binding and loop extrusion and could miss intermediate DNA loop extrusion steps by cohesin (22Kim Y. Shi Z. Zhang H. Finkelstein I.J. Yu H. Human cohesin compacts DNA by loop extrusion.Science. 2019; 366: 1345-1349Crossref PubMed Scopus (347) Google Scholar). Hence, because of technical challenges in studying dynamic multisubunit cohesin–NIPBL complexes, the mechanism of DNA binding and loop extrusion by cohesin is still under intense debate (25Bauer B.W. Davidson I.F. Canena D. Wutz G. Tang W. Litos G. et al.Cohesin mediates DNA loop extrusion by a "swing and clamp" mechanism.Cell. 2021; 184: 5448-5464.e5422Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 26Higashi T.L. Pobegalov G. Tang M. Molodtsov M.I. Uhlmann F. A Brownian ratchet model for DNA loop extrusion by the cohesin complex.Elife. 2021; 10: e67530Crossref PubMed Scopus (35) Google Scholar, 33Ryu J.K. Katan A.J. van der Sluis E.O. Wisse T. de Groot R. Haering C.H. et al.The condensin holocomplex cycles dynamically between open and collapsed states.Nat. Struct. Mol. Biol. 2020; 27: 1134-1141Crossref PubMed Scopus (40) Google Scholar, 34Nichols M.H. Corces V.G. A tethered-inchworm model of SMC DNA translocation.Nat. Struct. Mol. Biol. 2018; 25: 906-910Crossref PubMed Scopus (31) Google Scholar, 35Marko J.F. De Los Rios P. Barducci A. Gruber S. DNA-segment-capture model for loop extrusion by structural maintenance of chromosome (SMC) protein complexes.Nucleic Acids Res. 2019; 47: 6956-6972Crossref PubMed Scopus (59) Google Scholar, 36Higashi T.L. Uhlmann F. SMC complexes: lifting the lid on loop extrusion.Curr. Opin. Cell Biol. 2022; 74: 13-22Crossref PubMed Scopus (16) Google Scholar). Several key questions remain unanswered regarding DNA binding and loop extrusion by cohesin-NIPBL, such as the following: (1) How do each DNA-binding site and protein conformational change contribute to initial DNA binding and loop extrusion? (2) What sequential steps lead to DNA binding and initiation of a DNA loop? (3) What are the DNA loop extrusion step sizes? Here, we applied traditional AFM imaging in air and HS-AFM imaging in liquids to reveal the structure and dynamics of cohesin-NIPBL–mediated DNA binding and loop extrusion. Our AFM studies show that cohesin-NIPBL uses arm extension to capture DNA and initiate DNA loops independent of ATPase hydrolysis. Surprisingly, foot-like protrusions on cohesin-NIPBL can transiently bind to DNA and facilitate the loading of the cohesin–NIPBL complex onto DNA. Furthermore, HS-AFM imaging reveals distinct forward and reverse DNA loop extrusion steps. These results shed new light on the cohesin-mediated DNA loop extrusion mechanism and provide new directions for future investigation of diverse biological functions of cohesin. Recent studies demonstrated that cohesin-NIPBL contains multiple DNA-binding sites, including the ones on the interface between SMC1 and SMC3 hinges, SMC heads, SA1/SA2 (37Lin J. Countryman P. Chen H. Pan H. Fan Y. Jiang Y. et al.Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing.Nucleic Acids Res. 2016; 44: 6363-6376Crossref PubMed Scopus (25) Google Scholar), and NIPBL (25Bauer B.W. Davidson I.F. Canena D. Wutz G. Tang W. Litos G. et al.Cohesin mediates DNA loop extrusion by a "swing and clamp" mechanism.Cell. 2021; 184: 5448-5464.e5422Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). These DNA-binding sites are essential for DNA loop extrusion (25Bauer B.W. Davidson I.F. Canena D. Wutz G. Tang W. Litos G. et al.Cohesin mediates DNA loop extrusion by a "swing and clamp" mechanism.Cell. 2021; 184: 5448-5464.e5422Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Despite these new discoveries, our understanding of how each DNA-binding domain on cohesin-NIPBL contributes to cohesin loading onto DNA is limited. To directly address this question, we purified WT cohesinSA1-NIPBLc (Fig. 1B) (22Kim Y. Shi Z. Zhang H. Finkelstein I.J. Yu H. Human cohesin compacts DNA by loop extrusion.Science. 2019; 366: 1345-1349Crossref PubMed Scopus (347) Google Scholar, 38Shi Z. Gao H. Bai X.C. Yu H. Cryo-EM structure of the human cohesin-NIPBL-DNA complex.Science. 2020; 368: 1454-1459Crossref PubMed Scopus (106) Google Scholar), which was shown to be active in DNA loop extrusion and contains SA1 and the C-terminal HEAT repeat domain of NIPBL (22Kim Y. Shi Z. Zhang H. Finkelstein I.J. Yu H. Human cohesin compacts DNA by loop extrusion.Science. 2019; 366: 1345-1349Crossref PubMed Scopus (347) Google Scholar). We applied AFM imaging in air and HS-AFM imaging in liquids (39Ando T. High-speed atomic force microscopy.Curr. Opin. Chem. Biol. 2019; 51: 105-112Crossref PubMed Scopus (51) Google Scholar) to investigate the structure and dynamics of cohesinSA1-NIPBLc alone and in complexes with DNA. Consistent with the previous literature (25Bauer B.W. Davidson I.F. Canena D. Wutz G. Tang W. Litos G. et al.Cohesin mediates DNA loop extrusion by a "swing and clamp" mechanism.Cell. 2021; 184: 5448-5464.e5422Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), AFM images of the cohesinSA1-NIPBLc collected in the air (+2.5 mM ATP, Fig. 1C) showed monomers with SMC arms (blue arrows, Fig. 1C) distinguishable from the globular domain, that is, the lower compartment that includes SMC heads, RAD21, SA1, and NIPBLc. Based on their distinct arm features, cohesinSA1-NIPBLc monomers (Ntotal = 127) can be categorized into several classes (Fig. 1C), including closed-ring (18.1%), I-shape with closely aligned SMC arms (23.8%), open-arm (21.3%), and those unclassifiable (36.8%). These data suggest that the SMC1/SMC3 hinge interface is highly dynamic, switching between open-arm and closed-ring conformations. Importantly, hinge opening is consistent with the recent discovery of the SMC1/SMC3 hinge interface as one of the DNA entry gates for yeast cohesin (40Collier J.E. Nasmyth K.A. DNA passes through cohesin's hinge as well as its Smc3-kleisin interface.Elife. 2022; 11: e80310Crossref PubMed Google Scholar). Unexpectedly, in addition to arms, a subpopulation of WT cohesinSA1-NIPBLc molecules (∼40% to 65% from three protein preparations) showed short protrusions (feet). Among cohesinSA1-NIPBLc molecules showing the foot structure, approximately 34.0% displayed one foot and 66.0% displayed two feet (Fig. 1C). Relative to arms, the feet were positioned at the opposite side of the globular domain/lower compartment and displayed shorter lengths (25 nm ± 7 nm, N = 50) than the SMC1/SMC3 arms (51 nm ± 15 nm, N = 50). We hypothesized that each cohesinSA1–NIPBLc complex contains two feet, with the possibility of either one or two feet hidden under the globular domain in AFM images. We speculated that the foot structures are the C-terminal domains of SA1 and NIPBL, which were disordered in the cryo-EM structure of cohesinSA1-NIPBLc (38Shi Z. Gao H. Bai X.C. Yu H. Cryo-EM structure of the human cohesin-NIPBL-DNA complex.Science. 2020; 368: 1454-1459Crossref PubMed Scopus (106) Google Scholar). To test this hypothesis, we imaged five additional complexes, including cohesin-NIPBLc no SA1, cohesinSA1 no NIPBLc, cohesinSA1dc-NIPBL (containing SA1 1–1054 AAs without its C terminus), cohesinSA1-NIPBLdc (containing NIPBL1163-2603 AAs without its C terminus), and cohesinSA1dc-NIPBLdc. In AFM images, cohesin-NIPBLc no SA1, cohesinSA1 alone no NIPBLc, cohesinSA1dc-NIPBL, and cohesinSA1-NIPBLdc all displayed predominantly one foot (Fig. 1, D–G). We speculated that the small percentage of cohesin complexes showing two feet without either the SA1/NIPBL subunit or their C-terminal domains might be due to SA1 and NIPBL self-dimerization. Consistent with this hypothesis, while most of SA1dc existed as monomers, a small percentage of molecules displayed AFM volumes greater than SA1dc monomers (Fig. S1, A and B). For NIPBLdc alone, while the formation of large protein aggregations (∼50% of the total complexes) complicated the interpretation of the results, AFM image analysis also showed complexes displayed AFM volumes greater than NIPBL monomers (Fig. S1C). While the biological relevance of higher-order SA1 and NIPBL oligomers is unknown, these results suggest that cohesinSA1dc-NIPBL and cohesinSA1-NIPBLdc showing two feet could be due to the oligomerization of NIPBL and SA1, respectively, in a small population of cohesin complexes in vitro. Due to the aggregation of NIPBLdc alone, for cohesinSA1dc-NIPBLdc, we analyzed complexes with globular domain AFM volumes consistent with monomers, based on a previously established standard curve relating AFM volume and molecular weight (41Kaur P. Wu D. Lin J. Countryman P. Bradford K.C. Erie D.A. et al.Enhanced electrostatic force microscopy reveals higher-order DNA looping mediated by the telomeric protein TRF2.Sci. Rep. 2016; 620513Crossref Scopus (25) Google Scholar). This analysis revealed that cohesinSA1dc-NIPBLdc predominantly (∼95%, N = 70) showed no foot (Fig. 1H). The small percentages of cohesinSA1dc-NIPBLdc molecules showing either one (4%) or two (1%) additional protrusions might be due to arms from oligomerized complexes. In summary, AFM imaging in air shows that foot structures are distinct from SMC1/SMC3 arms and suggests that each C-terminal domain of SA1 and NIPBL contributes to one foot. A recent study identified three dsDNA-binding patches on SA1, including Patch 1 (K92, K95, K172, and K173), 2 (K555, K558, and R564), and 3 (K969, R971, K1013, and R1016) (25Bauer B.W. Davidson I.F. Canena D. Wutz G. Tang W. Litos G. et al.Cohesin mediates DNA loop extrusion by a "swing and clamp" mechanism.Cell. 2021; 184: 5448-5464.e5422Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). However, DNA binding by the C-terminal domains of SA1/SA2 and NIPBL, which were disordered in the cryo-EM structure of cohesin-NIPBL (38Shi Z. Gao H. Bai X.C. Yu H. Cryo-EM structure of the human cohesin-NIPBL-DNA complex.Science. 2020; 368: 1454-1459Crossref PubMed Scopus (106) Google Scholar), has not been investigated. SA1 and SA2 are highly similar, with approximately 70% sequence identity (42Kong X. Ball Jr., A.R. Pham H.X. Zeng W. Chen H.Y. Schmiesing J.A. et al.Distinct functions of human cohesin-SA1 and cohesin-SA2 in double-strand break repair.Mol. Cell. Biol. 2014; 34: 685-698Crossref PubMed Scopus (61) Google Scholar). To further establish DNA-binding domains on SA1/SA2, we purified WT full-length SA2 (1–1231 AAs) and SA2 fragments, including the N-terminal (1–301 AAs or 1–450 AAs) and C-terminal (1052–1231 AAs) domains (Fig. S2A) (43Zhang 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; 8e69458Google Scholar). Fluorescence anisotropy measurements using a fluorescently labeled dsDNA substrate (45 bp) revealed that SA2 contains extensive DNA-binding surfaces. Compared to the full-length SA2 (Kd = 63.5 nM ± 1.1 nM), the highest binding affinity is contributed by its N-terminal domain (1–302 AAs: Kd = 110.2 nM ± 7.1 nM; 1–450 AAs: Kd = 55.7 nM ± 0.4 nM) and its C-terminal domain binds to dsDNA weakly (1052–1231 AAs: Kd = 1500.2 nM ± 0.02 nM, Fig. S2, B–E). Consistent with these results, we showed previously that deletion of the C-terminal domain of SA2 reduces its binding affinity for dsDNA (44Countryman P. Fan Y. Gorthi A. Pan H. Strickland J. Kaur P. 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 (30) Google Scholar). Thus, these results from fluorescence anisotropy suggest that the C-terminal domain of SA1/SA2 has the potential to bind DNA. Indeed, the C-terminal domain of SA1/SA2 can easily get cleaved during protein purification (43Zhang 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; 8e69458Google Scholar), suggesting that this domain has an extended structure, consistent with the foot-like feature observed in AFM images. To study DNA binding by cohesin-NIPBL, we first employed AFM imaging in air to examine samples of cohesinSA1-NIPBLc and dsDNA (5.19 kb) deposited onto a mica surface (+2.5 mM ATP). Furthermore, to determine if ATPase activity changes DNA-binding modes, we purified the ATP-binding proficient and ATPase-deficient SMC1A-E1157Q/SMC3-E1144Q (EQ) cohesinSA1-NIPBLc mutant. Both WT and ATPase mutant cohesinSA1–NIPBLc complexes were randomly distributed on internal sites along dsDNA (Fig. S3). AFM images revealed different DNA-binding modes by WT cohesinSA1-NIPBLc, as seen previously for condensin (33Ryu J.K. Katan A.J. van der Sluis E.O. Wisse T. de Groot R. Haering C.H. et al.The condensin holocomplex cycles dynamically between open and collapsed states.Nat. Struct. Mol. Biol. 2020; 27: 1134-1141Crossref PubMed Scopus (40) Google Scholar). WT cohesinSA1-NIPBLc molecules bound to DNA through the arm-hinge (Fig. 2A, 30.0% ± 4.1%), the globular domain (53.0% ± 2.1%), both the arm-hinge and globular domains (15.0% ± 2.2%), or the foot (1.9% ± 0.2%). We observed similar DNA-binding modes by the ATPase-deficient EQ cohesinSA1-NIPBLc mutant in AFM images (+ATP, Fig. 2B). These results suggest that ATP hydrolysis is not needed for cohesinSA1-NIPBLc loading onto DNA. It is worth noting that a previous AFM study reported DNA binding through the globular and hinge domains of condensin (33Ryu J.K. Katan A.J. van der Sluis E.O. Wisse T. de Groot R. Haering C.H. et al.The condensin holocomplex cycles dynamically between open and collapsed states.Nat. Struct. Mol. Biol. 2020; 27: 1134-1141Crossref PubMed Scopus (40) Google Scholar). To further study how cohesin-NIPBL dynamically loads onto DNA and initiates a DNA loop, we applied HS-AFM imaging of WT or ATPase mutant cohesinSA1-NIPBLc in the presence of dsDNA. We recently developed robust sample deposition conditions on a 1-(3-Aminopropyl)silatrane (APS)-treated mica (APS-mica) surface (45Shlyakhtenko L.S. Gall A.A. Lyubchenko Y.L. Mica functionalization for imaging of DNA and protein-DNA complexes with atomic force microscopy.Methods Mol. Biol. 2013; 931: 295-312Crossref PubMed Google Scholar). This development enabled us to observe real-time domain protrusion by Twinkle helicase during initial DNA loading (46Li Z. Kaur P. Lo C.Y. Chopra N. Smith J. Wang H. et al.Structural and dynamic basis of DNA capture and translocation by mitochondrial Twinkle helicase.Nucleic Acids Res. 2022; 50: 11965-11978Crossref PubMed Scopus (3) Google Scholar). We first deposited WT cohesinSA1-NIPBLc (30 nM) with DNA (3 nM, 5.19 kb) onto an APS-mica surface after 16-fold dilution and scanned the sample in a buffer containing ATP (+4 mM ATP) using either a Cypher VRS or JPK NanoWizard HS-AFM at a scan rate of 0.4 to 2.3 frames/s. Importantly, under our sample deposition and imaging conditions, both proteins and DNA were mobile on the APS-mica surface. In time-lapse HS-AFM images, cohesinSA1-NIPBLc displayed similar conformations as observed in the static images collected in air (Fig. 1), including I-shape, closed-ring, and folded-arm with some complexes showing protruding feet (Fig. 3A). CohesinSA1-NIPBLc was highly dynamic in the presence of DNA (Fig. 3B). Figure 3B shows one example of a monomeric WT cohesinSA1-NIPBLc molecule with two arms and a bent elbow extending it