@article{irvin_wang_2024, title={Single-molecule fluorescence imaging of DNA maintenance protein binding dynamics and activities on extended DNA}, volume={87}, ISSN={["1879-033X"]}, DOI={10.1016/j.sbi.2024.102863}, abstractNote={Defining the molecular mechanisms by which genome maintenance proteins dynamically associate with and process DNA is essential to understand the potential avenues by which these stabilizing mechanisms are disrupted. Single-molecule fluorescence imaging (SMFI) of protein dynamics on extended DNA has greatly expanded our ability to accomplish this, as it captures singular biomolecular interactions - in all their complexity and diversity - without relying on ensemble-averaging of bulk protein activity as most traditional biochemical techniques must do. In this review, we discuss how SMFI studies with extended DNA have substantially contributed to genome stability research over the past two years.}, journal={CURRENT OPINION IN STRUCTURAL BIOLOGY}, author={Irvin, Elizabeth Marie and Wang, Hong}, year={2024}, month={Aug} }
 @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. 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. 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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. 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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}, 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{irvin_wang_2023, title={Single-molecule imaging of genome maintenance proteins encountering specific DNA sequences and structures}, volume={128}, ISSN={["1568-7856"]}, DOI={10.1016/j.dnarep.2023.103528}, abstractNote={DNA repair pathways are tightly regulated processes that recognize specific hallmarks of DNA damage and coordinate lesion repair through discrete mechanisms, all within the context of a three-dimensional chromatin landscape. Dysregulation or malfunction of any one of the protein constituents in these pathways can contribute to aging and a variety of diseases. While the collective action of these many proteins is what drives DNA repair on the organismal scale, it is the interactions between individual proteins and DNA that facilitate each step of these pathways. In much the same way that ensemble biochemical techniques have characterized the various steps of DNA repair pathways, single-molecule imaging (SMI) approaches zoom in further, characterizing the individual protein-DNA interactions that compose each pathway step. SMI techniques offer the high resolving power needed to characterize the molecular structure and functional dynamics of individual biological interactions on the nanoscale. In this review, we highlight how our lab has used SMI techniques – traditional atomic force microscopy (AFM) imaging in air, high-speed AFM (HS-AFM) in liquids, and the DNA tightrope assay – over the past decade to study protein-nucleic acid interactions involved in DNA repair, mitochondrial DNA replication, and telomere maintenance. We discuss how DNA substrates containing specific DNA sequences or structures that emulate DNA repair intermediates or telomeres were generated and validated. For each highlighted project, we discuss novel findings made possible by the spatial and temporal resolution offered by these SMI techniques and unique DNA substrates.}, journal={DNA REPAIR}, author={Irvin, Elizabeth Marie and Wang, Hong}, year={2023}, month={Aug} }
 @article{leighton_irvin_kaur_liu_you_bhattaram_piehler_riehn_wang_pan_et al._2022, title={Densely methylated DNA traps Methyl-CpG-binding domain protein 2 but permits free diffusion by Methyl-CpG-binding domain protein 3}, volume={298}, ISSN={["1083-351X"]}, url={https://doi.org/10.1016/j.jbc.2022.102428}, DOI={10.1016/j.jbc.2022.102428}, abstractNote={The methyl-CpG–binding domain 2 and 3 proteins (MBD2 and MBD3) provide structural and DNA-binding function for the Nucleosome Remodeling and Deacetylase (NuRD) complex. The two proteins form distinct NuRD complexes and show different binding affinity and selectivity for methylated DNA. Previous studies have shown that MBD2 binds with high affinity and selectivity for a single methylated CpG dinucleotide while MBD3 does not. However, the NuRD complex functions in regions of the genome that contain many CpG dinucleotides (CpG islands). Therefore, in this work, we investigate the binding and diffusion of MBD2 and MBD3 on more biologically relevant DNA templates that contain a large CpG island or limited CpG sites. Using a combination of single-molecule and biophysical analyses, we show that both MBD2 and MBD3 diffuse freely and rapidly across unmethylated CpG-rich DNA. In contrast, we found methylation of large CpG islands traps MBD2 leading to stable and apparently static binding on the CpG island while MBD3 continues to diffuse freely. In addition, we demonstrate both proteins bend DNA, which is augmented by methylation. Together, these studies support a model in which MBD2-NuRD strongly localizes to and compacts methylated CpG islands while MBD3-NuRD can freely mobilize nucleosomes independent of methylation status. The methyl-CpG–binding domain 2 and 3 proteins (MBD2 and MBD3) provide structural and DNA-binding function for the Nucleosome Remodeling and Deacetylase (NuRD) complex. The two proteins form distinct NuRD complexes and show different binding affinity and selectivity for methylated DNA. Previous studies have shown that MBD2 binds with high affinity and selectivity for a single methylated CpG dinucleotide while MBD3 does not. However, the NuRD complex functions in regions of the genome that contain many CpG dinucleotides (CpG islands). Therefore, in this work, we investigate the binding and diffusion of MBD2 and MBD3 on more biologically relevant DNA templates that contain a large CpG island or limited CpG sites. Using a combination of single-molecule and biophysical analyses, we show that both MBD2 and MBD3 diffuse freely and rapidly across unmethylated CpG-rich DNA. In contrast, we found methylation of large CpG islands traps MBD2 leading to stable and apparently static binding on the CpG island while MBD3 continues to diffuse freely. In addition, we demonstrate both proteins bend DNA, which is augmented by methylation. Together, these studies support a model in which MBD2-NuRD strongly localizes to and compacts methylated CpG islands while MBD3-NuRD can freely mobilize nucleosomes independent of methylation status. The methyl-CpG–binding domain (MBD) family of proteins binds methylated DNA through a conserved domain that recognizes the symmetrically related methylcytosines in a cytosine-guanosine dinucleotide (CpG) (1Hendrich B. Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins.Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1089) Google Scholar). The structure of this domain bound to a single methylated CpG (mCpG) site has been determined for most members of the MBD family (2Ohki I. Shimotake N. Fujita N. Jee J. Ikegami T. Nakao M. et al.Solution structure of the methyl-CpG binding domain of human MBD1 in complex with methylated DNA.Cell. 2001; 105: 487-497Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 3Ho K.L. 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Genet. 2009; 41: 1350-1353Crossref PubMed Scopus (965) Google Scholar). Furthermore, methylation of CpG islands in promoters and enhancers correlates with nucleosome occupancy, chromatin compaction, and associated gene silencing. Hence, we have investigated how MBD proteins bind and diffuse along these CpG islands to better understand the functional consequences in a more biologically relevant context. In the current work, we focus on the structure and dynamics of the MBD2 and MBD3 proteins. These two highly homologous proteins arose from a duplication of the ancestral MBD present across the animal kingdom (1Hendrich B. Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins.Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1089) Google Scholar, 15Cramer J.M. Pohlmann D. Gomez F. Mark L. Kornegay B. Hall C. et al.Methylation specific targeting of a chromatin remodeling complex from sponges to humans.Sci. 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Karuppasamy M. Basu S. Murthy A.S. et al.The nucleosome remodeling and deacetylase complex NuRD is built from preformed catalytically active sub-modules.J. Mol. Biol. 2016; 428: 2931-2942Crossref PubMed Scopus (47) Google Scholar, 20Zhang Y. Ng H.H. Erdjument-Bromage H. Tempst P. Bird A. Reinberg D. Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation.Genes Dev. 1999; 13: 1924-1935Crossref PubMed Scopus (937) Google Scholar) consists of a least six additional proteins, each of which has multiple paralogs that provide histone deacetylase activity (HDAC1/2), histone binding, and chromatin remodeling function (CHD3/4), and protein–protein interactions (GATAD2A/B, RBBP4/7, MTA1/2/3, CDK2AP1). The MBD2 and MBD3 proteins form distinct NuRD complexes that appear to have unique functional roles (16Le Guezennec X. Vermeulen M. Brinkman A.B. Hoeijmakers W.A. Cohen A. Lasonder E. et al.MBD2/NuRD and MBD3/NuRD, two distinct complexes with different biochemical and functional properties.Mol. Cell. Biol. 2006; 26: 843-851Crossref PubMed Scopus (266) Google Scholar, 21Leighton G. Williams Jr., D.C. The methyl-CpG-binding domain 2 and 3 proteins and formation of the nucleosome remodeling and deacetylase complex.J. Mol. Biol. 2020; 432: 1624-1639Crossref Scopus (18) Google Scholar, 22Yu X. Azzo A. Bilinovich S.M. Li X. Dozmorov M. Kurita R. et al.Disruption of the MBD2-NuRD complex but not MBD3-NuRD induces high level HbF expression in human adult erythroid cells.Haematologica. 2019; 104: 2361-2371Crossref PubMed Scopus (32) Google Scholar). The two proteins show different levels of selectivity for mCpGs attributable primarily to a single amino acid change from tyrosine (MBD2) to phenylalanine (MBD3) within the DNA-binding site (Fig. 1) (1Hendrich B. Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins.Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1089) Google Scholar, 6Cramer J.M. Scarsdale J.N. Walavalkar N.M. Buchwald W.A. Ginder G.D. Williams Jr., D.C. Probing the dynamic distribution of bound states for methylcytosine-binding domains on DNA.J. Biol. Chem. 2014; 289: 1294-1302Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 23Baubec T. Ivanek R. Lienert F. Schubeler D. Methylation-dependent and -independent genomic targeting principles of the MBD protein family.Cell. 2013; 153: 480-492Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 24Saito M. Ishikawa F. The mCpG-binding domain of human MBD3 does not bind to mCpG but interacts with NuRD/Mi2 components HDAC1 and MTA2.J. Biol. Chem. 2002; 277: 35434-35439Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). MBD2 shows up to 100-fold selectivity for a fully mCpG dinucleotide compared to an unmethylated CpG (1Hendrich B. Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins.Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1089) Google Scholar, 4Scarsdale J.N. Webb H.D. Ginder G.D. Williams Jr., D.C. Solution structure and dynamic analysis of chicken MBD2 methyl binding domain bound to a target-methylated DNA sequence.Nucleic Acids Res. 2011; 39: 6741-6752Crossref PubMed Scopus (80) Google Scholar, 25Desai M.A. Webb H.D. Sinanan L.M. Scarsdale J.N. Walavalkar N.M. Ginder G.D. et al.An intrinsically disordered region of methyl-CpG binding domain protein 2 (MBD2) recruits the histone deacetylase core of the NuRD complex.Nucleic Acids Res. 2015; 43: 3100-3113Crossref PubMed Scopus (44) Google Scholar, 26Hashimoto H. Liu Y. Upadhyay A.K. Chang Y. Howerton S.B. Vertino P.M. et al.Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation.Nucleic Acids Res. 2012; 40: 4841-4849Crossref PubMed Scopus (356) Google Scholar). In contrast, MBD3 binds DNA with an overall much lower affinity and shows no or slight (3–5 fold) selectivity for an mCpG (6Cramer J.M. Scarsdale J.N. Walavalkar N.M. Buchwald W.A. Ginder G.D. Williams Jr., D.C. Probing the dynamic distribution of bound states for methylcytosine-binding domains on DNA.J. Biol. Chem. 2014; 289: 1294-1302Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 26Hashimoto H. Liu Y. Upadhyay A.K. Chang Y. Howerton S.B. Vertino P.M. et al.Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation.Nucleic Acids Res. 2012; 40: 4841-4849Crossref PubMed Scopus (356) Google Scholar). Consistent with this binding difference, previous genomic localization studies found that MBD2-NuRD predominantly binds at heavily mCpG islands associated with silenced genes, whereas MBD3 localizes to methylated and unmethylated CpG islands associated with expressed genes (23Baubec T. Ivanek R. Lienert F. Schubeler D. Methylation-dependent and -independent genomic targeting principles of the MBD protein family.Cell. 2013; 153: 480-492Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 27Menafra R. Brinkman A.B. Matarese F. Franci G. Bartels S.J. Nguyen L. et al.Genome-wide binding of MBD2 reveals strong preference for highly methylated loci.PLoS One. 2014; 9: e99603Crossref PubMed Scopus (37) Google Scholar, 28Shimbo T. Du Y. Grimm S.A. Dhasarathy A. Mav D. Shah R.R. et al.MBD3 localizes at promoters, gene bodies and enhancers of active genes.Plos Genet. 2013; 9e1004028Crossref PubMed Scopus (82) Google Scholar, 29Gunther K. Rust M. Leers J. Boettger T. Scharfe M. Jarek M. et al.Differential roles for MBD2 and MBD3 at methylated CpG islands, active promoters and binding to exon sequences.Nucleic Acids Res. 2013; 41: 3010-3021Crossref PubMed Scopus (90) Google Scholar). However, recent data suggests the alternative interpretation that both MBD2 and MBD3 depend on methylation for proper localization across the genome (30Hainer S.J. McCannell K.N. Yu J. Ee L.-S. Zhu L.J. Rando O.J. et al.DNA methylation directs genomic localization of Mbd2 and Mbd3 in embryonic stem cells.Elife. 2016; 5: e21964Crossref PubMed Scopus (21) Google Scholar). Therefore, how methylation selectivity of the MBD2 and MBD3 proteins impacts this localization and function remains an open question in the field. In previous work, we used single-molecule analyses to study the behavior of the isolated MBD from MBD2 on various DNA substrates (31Pan H. Bilinovich S.M. Kaur P. Riehn R. Wang H. Williams Jr., D.C. CpG and methylation-dependent DNA binding and dynamics of the methylcytosine binding domain 2 protein at the single-molecule level.Nucleic Acids Res. 2017; 45: 9164-9177Crossref PubMed Scopus (19) Google Scholar). Consistent with NMR and bulk biochemical studies, we found a remarkable difference in DNA bending and sliding of the MBD from MBD2 (MBD2MBD) on methylated and unmethylated DNA containing CpG islands. MBD2MBD is mostly restricted to the mCpG islands, while it freely diffuses across unmethylated CpG islands. Furthermore, we also uncovered a novel role for the intrinsically disordered region (IDR) of MBD2 in DNA bending (25Desai M.A. Webb H.D. Sinanan L.M. Scarsdale J.N. Walavalkar N.M. Ginder G.D. et al.An intrinsically disordered region of methyl-CpG binding domain protein 2 (MBD2) recruits the histone deacetylase core of the NuRD complex.Nucleic Acids Res. 2015; 43: 3100-3113Crossref PubMed Scopus (44) Google Scholar). The DNA-bending angle induced by both the MBD and a small portion of the IDR (MBD2MBD+IDR) on unmethylated CpG-rich DNA is larger than observed for CpG-free and further increases upon binding mCpG-rich DNA. In separate structural studies of MBD3, we found that the MBD from MBD3 shows only weak selectivity for a single mCpG within a small (17 bp) dsDNA fragment (6Cramer J.M. Scarsdale J.N. Walavalkar N.M. Buchwald W.A. Ginder G.D. Williams Jr., D.C. Probing the dynamic distribution of bound states for methylcytosine-binding domains on DNA.J. Biol. Chem. 2014; 289: 1294-1302Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Based on a combination of chemical shift analyses, mutagenesis, and residual dipolar coupling measurements, we showed that MBD3 exchanges rapidly between CpG-specific and nonspecific binding modes, leading to chemical shift averaging between these two states. Hence, MBD3 recognizes an mCpG site, as evidenced by significant chemical shift changes, but does not strongly localize to this site when bound to DNA. This difference between the DNA-binding dynamics of MBD2 and MBD3 correlates with the prior localization studies that show both MBD2 and MBD3 localize to unmethylated CpG islands, while MBD2 more exclusively localizes to mCpG islands (23Baubec T. Ivanek R. Lienert F. Schubeler D. Methylation-dependent and -independent genomic targeting principles of the MBD protein family.Cell. 2013; 153: 480-492Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 27Menafra R. Brinkman A.B. Matarese F. Franci G. Bartels S.J. Nguyen L. et al.Genome-wide binding of MBD2 reveals strong preference for highly methylated loci.PLoS One. 2014; 9: e99603Crossref PubMed Scopus (37) Google Scholar, 28Shimbo T. Du Y. Grimm S.A. Dhasarathy A. Mav D. Shah R.R. et al.MBD3 localizes at promoters, gene bodies and enhancers of active genes.Plos Genet. 2013; 9e1004028Crossref PubMed Scopus (82) Google Scholar, 29Gunther K. Rust M. Leers J. Boettger T. Scharfe M. Jarek M. et al.Differential roles for MBD2 and MBD3 at methylated CpG islands, active promoters and binding to exon sequences.Nucleic Acids Res. 2013; 41: 3010-3021Crossref PubMed Scopus (90) Google Scholar). Despite these recent studies, we do not know how the remaining, largely unstructured regions of MBD2 and MBD3 influence diffusion along DNA. Furthermore, it is unclear how reduced selectivity and binding affinity of MBD3 modifies its distribution and sliding on methylated and unmethylated CpG islands, which contain many CpG sites. To address these questions, in the current studies, we use a combination of biophysical techniques, including atomic force microscopy (AFM) imaging (32Benarroch-Popivker D. Pisano S. Mendez-Bermudez A. Lototska L. Kaur P. Bauwens S. et al.TRF2-Mediated control of telomere DNA topology as a mechanism for chromosome-end protection.Mol. Cell. 2016; 61: 274-286Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 33Wang H. Tessmer I. 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Chem. 2014; 289: 1294-1302Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) to measure the binding and sliding of MBD2 and MBD3 on methylated and unmethylated DNA substrates. In the DNA tightrope assay, DNA molecules are stretched under hydrodynamic flow inside a flow cell. Anchoring of stretched DNA between poly-L-lysine–coated silica microspheres leads to the formation of DNA tightropes at an elongation of ∼90% of the DNA contour length (Fig. 2A). The spatial resolution of the DNA tightrope assay was estimated to be 16 nm (37Lin J. Countryman P. Buncher N. Kaur P. E L. Zhang Y. et al.TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.Nucleic Acids Res. 2014; 42: 2493-2504Crossref PubMed Scopus (51) Google Scholar). Uniquely, DNA tightropes created using tandemly ligated DNA allow us to directly correlate DNA-binding events with the underlying specific DNA sequences or structures such as three-stranded R-loops (31Pan H. Bilinovich S.M. Kaur P. Riehn R. Wang H. Williams Jr., D.C. CpG and methylation-dependent DNA binding and dynamics of the methylcytosine binding domain 2 protein at the single-molecule level.Nucleic Acids Res. 2017; 45: 9164-9177Crossref PubMed Scopus (19) Google Scholar, 32Benarroch-Popivker D. Pisano S. Mendez-Bermudez A. Lototska L. Kaur P. Bauwens S. et al.TRF2-Mediated control of telomere DNA topology as a mechanism for chromosome-end protection.Mol. Cell. 2016; 61: 274-286Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 37Lin J. Countryman P. Buncher N. Kaur P. E L. Zhang Y. et al.TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.Nucleic Acids Res. 2014; 42: 2493-2504Crossref PubMed Scopus (51) Google Scholar, 38Pan H. Jin M. Ghadiyaram A. Kaur P. Miller H.E. Ta H.M. et al.Cohesin SA1 and SA2 are RNA binding proteins that localize to RNA containing regions on DNA.Nucleic Acids Res. 2020; 48: 5639-5655Crossref PubMed Google Scholar). To study MBD proteins diffusion, we ligated linear DNA fragments to form DNA tightropes with CpG free or alternating CpG-free and CpG-rich regions (Fig. 2B and S8A). The results from our previous study revealed that the isolated MBD from MBD2, with or without a small portion of the adjacent IDR (MBD2MBD and MBD2MBD+IDR), carry out unbiased 1D diffusion on CpG-rich DNA but undergoes subdiffusion on CpG-free DNA. In contrast, both proteins stably and statically bind to mCpG regions. In this study, we purified a construct that contains almost the entire length of the MBD2b isoform plus the coiled-coil region from GATAD2A (MBD2sc) to investigate how these additional regions impact DNA binding and diffusion (Experimental Procedures, Fig. 1A). We previously found that the MBD2sc binds DNA with an approximately 100× higher affinity than MBD2MBD (25Desai M.A. Webb H.D. Sinanan L.M. Scarsdale J.N. Walavalkar N.M. Ginder G.D. et al.An intrinsically disordered region of methyl-CpG binding domain protein 2 (MBD2) recruits the histone deacetylase core of the NuRD complex.Nucleic Acids Res. 2015; 43: 3100-3113Crossref PubMed Scopus (44) Google Scholar). Consistent with these results, fluorescence anisotropy experiments showed that MBD2sc binds to DNA containing unmethylated, methylated, or no CpG site with equilibrium dissociation constants of 2.8 (±0.1 μM), 0.007 (±0.002 μM), or 9.1 (±0.4 μM), respectively (Fig. 2C). Hence, we questioned whether the additional binding affinity provided by the IDR in MBD2 would modify DNA binding and sliding. We directly addressed this question using the DNA tightrope assay. For the DNA tightrope assay, we first generated DNA substrates by tandemly ligating linear DNA fragments containing CpG-free or unmethylated CpG-free–rich sequences (Fig. 2B). Further, we conjugated His-tagged MBD2sc to streptavidin-coated quantum dots (SAv-QDs) through the multivalent chelator tris-nitrilotriacetic acid linker (Fig. 2A) (39Reichel A. Schaible D. Al Furoukh N. Cohen M. Schreiber G. Piehler J. Noncovalent, site-specific biotinylation of histidine-tagged proteins.Anal. Chem. 2007; 79: 8590-8600Crossref PubMed Scopus (47) Google Scholar). Following the formation of the DNA tightropes, we introduced QD-labeled MBD2sc into the flow cell. Analysis of MBD2sc on DNA tightropes revealed two populations (Table 1): apparently immobile throughout data acquisition and mobile molecules (Fig. 2D). To exclude that this apparently immobile population reflects aggregation, we categorized particles as a single protein or cluster based on their individual QD blinking rate (Table S1) which shows that only a small fraction comprises multiple proteins. To obtain diffusion coefficients for mobile MBD2sc on DNA tightropes, we tracked the position of MBD2sc-QDs on DNA by Gaussian fitting to kymographs (particle position versus time plots) (36Kad N.M. Wang H. Kennedy G.G. Warshaw D.M. Van Houten B. Collaborative dynamic DNA scanning by nucleotide excision repair proteins investigated by single- molecule imaging of quantum-dot-labeled proteins.Mol. Cell. 2010; 37: 702-713Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 40Dunn A.R. Kad N.M. Nelson S.R. Warshaw D.M. Wallace S.S. Single Qdot-labeled glycosylase molecules use a wedge amino acid to probe for lesions while scanning along DNA.Nucleic Acids Res. 2011; 39: 7487-7498Crossref PubMed Scopus (98) Google Scholar). We obtained diffusion coefficients and alpha exponents by fitting the mean square displacement (MSD) versus time. An alpha exponent of 1 indicates an unbiased random walk, and a value less than 1 indicates subdiffusion (41Saxton M.J. Single-particle tracking: The distribution of diffusion coefficients.Biophys. J. 1997; 72: 1744-1753Abstract Full Text PDF PubMed Scopus (479) Google Scholar). The diffusion coefficients displayed by MBD2sc on the CpG-free DNA tightrope were significantly slower than those on DNA tightropes containing CpG sites (CpG-free–rich) (Fig. 2E). In addition, on the CpG-free–rich tightropes, MBD2sc displayed alpha exponents close to 1 (1.0 ± 0.2), indicating largely unbiased 1D diffusion on DNA (Fig. 2F, Table 2). As compared to CpG-free–rich DNA, the alpha exponents for MBD2sc on CpG-free DNA tightropes were slightly (p < 0.001) reduced (0.8 ± 0.2, Fig. 2F, Table 2). Overall, MBD2sc displayed slightly different diffusion ranges on CpG-free DNA tightropes compared to CpG-free–rich DNA tightropes containing multiple CpG sites (Fig. S3). In summary, MBD2sc shows more rapid and extensive 1D diffusion on CpG-free–rich DNA than CpG-free sequences.Table 1Fraction of statically bound MBD2sc and MBD3sc on unmethylated- and methylated-DNA tightropesDNAMBD2scMBD3scStatic binding (%)NStatic binding (%)NCpG-free–rich23 ± 614747 ± 7123CpG-free19 ± 633354 ± 8240mCpG-free–rich96 ± 324652 ± 10345mCpG-mini90 ± 149550 ± 8158The values represent mean ± SD from 2 to 3 experiments for each data set. Open table in a new tab Table 2Diffusion coefficient of MBD2sc and MBD3sc on different DNA substratesDNAMBD2scMBD3scD (μm2/s)α exponentND (μm2/s)α exponentNCpG-free0.04 ± 0.020.8 ± 0.2990.04 ± 0.030.8 ± 0.3199CpG-free–rich0.15 ± 0.051.0 ± 0.21000.04 ± 0.010.9 ± 0.2144mCpG-free–richNA.NA.0.09 ± 0.021.0 ± 0.2174mCpG-miniNA.NA.0.05 ± 0.020.9 ± 0.170The values represent mean ± SD from 2 to 4 experiments for each data set. Open table in a new tab The values represent mean ± SD from 2 to 3 experiments for each data set. The values represent mean ± SD from 2 to 4 experiments for each data set. To evaluate how DNA methylation affects the dynamics of MBD2sc on DNA, we imaged QD-labeled MBD2sc on the CpG-free–rich DNA tightropes after methylation. Linear DNA substrates were methylated before ligation using CpG Methyltransferase (M.SssI) with SAM as a cofactor (Experimental procedures). We confirmed methylation of the linear CpG-free–rich DNA substrate by digestion with the methylation-sensitive HpaII restriction endonuclease (Fig. S1). We then tandemly ligated the mCpG-free–rich (mCpG-free-rich, Fig. 3A) and used it to form DNA tightropes between silica beads. Compared to unmethylated CpG-free–rich and CpG-free DNA, the binding density of MBD2sc increased approximately 4× on mCpG-free–rich DNA tightropes (Fig. 3B, S4A). This result is consistent with the preferential binding of MBD2sc to mCpG sites, as supported by binding affinity measurements (Fig. 2C). Notably, while the majority of MBD2sc observed on unmethylated CpG-free–rich DNA tightropes was mobile (77%), on mCpG-free–rich tightropes, the majority of the protein bound was apparently immobile (96%) throughout data acquisition (Fig. 3B and Table 1). Furthermore, the distance between adjacent proteins on the mCpG-free–rich DNA tightropes is Gaussian distributed, with the peak centered at 2.3 (±0.3 μm) (Fig. 3C). This spacing matches the calculated distances between adjacent mCpG-rich regions on DNA tightropes (Fig. 3A), considering that they are stretched to ∼90% of their contour length. Taken together, fluorescence imaging of MBD2sc on DNA tightropes establishes that MBD2sc recognizes mCpG islands through stable and apparently static binding. To evaluate whether MBD2s}, number={10}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Leighton, Gage O. and Irvin, Elizabeth Marie and Kaur, Parminder and Liu, Ming and You, Changjiang and Bhattaram, Dhruv and Piehler, Jacob and Riehn, Robert and Wang, Hong and Pan, Hai and et al.}, year={2022}, month={Oct} }