{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,12]],"date-time":"2026-09-12T02:08:39Z","timestamp":1789178919987,"version":"build-2803163510"},"posted":{"date-parts":[[2026,9,8]]},"group-title":"Biophysics","reference-count":61,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T00:00:00Z","timestamp":1788825600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/publicdomain\/zero\/1.0\/"}],"funder":[{"award":["1ZIAHL001056"],"award-info":[{"award-number":["1ZIAHL001056"]}],"id":[{"id":"https:\/\/ror.org\/023ny1p48","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,8]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Type II topoisomerases (topo IIs) are essential enzymes that regulate DNA topology through a strand-passage mechanism in which a duplex DNA (transfer-segment) is passed through a transiently cleaved second duplex DNA (gate-segment). Biochemical and structural approaches have revealed critical details of the binding and cleavage of the gate-segment DNA. However, capture of the transfer-segment DNA has proven more difficult to resolve due to the transient nature of the interaction. Nonetheless, selection of the transfer segment with a specific conformation or orientation relative to the gate segment is predicted to govern aspects of topo II activity, including chiral discrimination and the ability to reduce topological complexity below equilibrium. To determine the conformation and orientation of the transfer-segment relative to the gate segment, we combined experimental single-molecule measurements of topo II unlinking a single DNA crossing with Brownian dynamics simulations of the DNA crossing. By correlating the unlinking rate with the geometric features of the DNA crossing, we obtain the complete three-dimensional preferred crossing geometry for strand passage. Strikingly, the preferred crossing geometries for\n                  <jats:italic>Escherichia coli<\/jats:italic>\n                  topoisomerase IV and\n                  <jats:italic>Methanosarcina mazei<\/jats:italic>\n                  topoisomerase VI are distinct and provide structural models of the DNA synapse selected for strand passage along with a mechanistic basis for their differing activities and biological functions. The approach we develop is generalizable, providing unique insights into the kinetic selection of DNA synapse structure.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Significance Statement<\/jats:title>\n                  <jats:p>Type II topoisomerases resolve DNA entanglements by passing one DNA duplex through a transient break in another, but how these enzymes select the three-dimensional geometry of the DNA segments they act on has remained unresolved because the interaction is too transient to capture structurally. We developed an approach combining single-molecule measurements with Brownian dynamics simulations to determine the crossing geometry each enzyme kinetically selects. Applied to two distinct topoisomerases, this approach revealed different preferred geometries that explain long-standing differences in how these enzymes sense DNA handedness and challenge a leading model for how topoisomerases simplify DNA tangles beyond what thermodynamics alone would predict. This kinetics-based strategy is broadly applicable to other enzymes that resolve DNA or RNA synapses.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.09.04.749274","type":"posted-content","created":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T04:20:17Z","timestamp":1788927617000},"source":"Crossref","is-referenced-by-count":0,"title":["Type II topoisomerase substrate geometry revealed through combined experiment and computation"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0524-7523","authenticated-orcid":false,"given":"Mihirkumar N.","family":"Prajapati","sequence":"first","affiliation":[{"name":"Department of Mechanical Engineering, University of Maryland, College Park, MD 20742, USA"},{"name":"Laboratory of Single Molecule Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yeonee","family":"Seol","sequence":"additional","affiliation":[{"name":"Laboratory of Single Molecule Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jonathan","family":"Silver","sequence":"additional","affiliation":[{"name":"Laboratory of Single Molecule Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3905-3471","authenticated-orcid":false,"given":"Parth R.","family":"Desai","sequence":"additional","affiliation":[{"name":"Department of Mechanical Engineering, University of Maryland, College Park, MD 20742, USA"},{"name":"Laboratory of Single Molecule Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA"},{"name":"Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Siddhartha","family":"Das","sequence":"additional","affiliation":[{"name":"Department of Mechanical Engineering, University of Maryland, College Park, MD 20742, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0863-5671","authenticated-orcid":false,"given":"Keir C.","family":"Neuman","sequence":"additional","affiliation":[{"name":"Laboratory of Single Molecule Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026091118350951000_2026.09.04.749274v1.1","doi-asserted-by":"publisher","DOI":"10.1002\/bies.202000286"},{"key":"2026091118350951000_2026.09.04.749274v1.2","doi-asserted-by":"crossref","unstructured":"N. G. Bush , K. Evans-Roberts , A. Maxwell , DNA Topoisomerases. EcoSal Plus 6 (2015).","DOI":"10.1128\/ecosalplus.esp-0010-2014"},{"key":"2026091118350951000_2026.09.04.749274v1.3","doi-asserted-by":"publisher","DOI":"10.32607\/actanaturae.11058"},{"key":"2026091118350951000_2026.09.04.749274v1.4","doi-asserted-by":"publisher","DOI":"10.1017\/S0033583598003424"},{"key":"2026091118350951000_2026.09.04.749274v1.5","doi-asserted-by":"crossref","unstructured":"J. A. Nickoloff , M. F. Hoekstra , DNA Damage and Repair: Advances from phage to humans (Springer Science & Business Media, 2001).","DOI":"10.1007\/978-1-59259-095-7"},{"key":"2026091118350951000_2026.09.04.749274v1.6","doi-asserted-by":"publisher","DOI":"10.1038\/s41580-022-00452-3"},{"key":"2026091118350951000_2026.09.04.749274v1.7","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2008721117"},{"key":"2026091118350951000_2026.09.04.749274v1.8","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2114024119"},{"key":"2026091118350951000_2026.09.04.749274v1.9","doi-asserted-by":"crossref","first-page":"9777","DOI":"10.1093\/nar\/gkae638","article-title":"Highly sensitive mapping of in vitro type II topoisomerase DNA cleavage sites with SHAN-seq","volume":"52","year":"2024","journal-title":"Nucleic Acids Res"},{"key":"2026091118350951000_2026.09.04.749274v1.10","doi-asserted-by":"publisher","DOI":"10.1017\/S003358350800468X"},{"key":"2026091118350951000_2026.09.04.749274v1.11","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.bi.65.070196.003223"},{"key":"2026091118350951000_2026.09.04.749274v1.12","doi-asserted-by":"publisher","DOI":"10.1021\/bi700789g"},{"key":"2026091118350951000_2026.09.04.749274v1.13","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkr258"},{"key":"2026091118350951000_2026.09.04.749274v1.14","doi-asserted-by":"publisher","DOI":"10.1002\/bies.10245"},{"key":"2026091118350951000_2026.09.04.749274v1.15","doi-asserted-by":"publisher","DOI":"10.1126\/science.277.5326.690"},{"key":"2026091118350951000_2026.09.04.749274v1.16","doi-asserted-by":"publisher","DOI":"10.1038\/nsmb1264"},{"key":"2026091118350951000_2026.09.04.749274v1.17","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2008.11.056"},{"key":"2026091118350951000_2026.09.04.749274v1.18","doi-asserted-by":"publisher","DOI":"10.1016\/S0022-2836(02)00447-3"},{"key":"2026091118350951000_2026.09.04.749274v1.19","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkp250"},{"key":"2026091118350951000_2026.09.04.749274v1.20","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.061029098"},{"key":"2026091118350951000_2026.09.04.749274v1.21","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkr109"},{"key":"2026091118350951000_2026.09.04.749274v1.22","doi-asserted-by":"publisher","DOI":"10.1038\/nature06396"},{"key":"2026091118350951000_2026.09.04.749274v1.23","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkt238"},{"key":"2026091118350951000_2026.09.04.749274v1.24","doi-asserted-by":"publisher","DOI":"10.1126\/science.adl5899"},{"key":"2026091118350951000_2026.09.04.749274v1.25","doi-asserted-by":"crossref","unstructured":"E. Michalczyk et al., Structural basis of chiral wrap and T-segment capture by Escherichia coli DNA gyrase. Proc. Natl. Acad. Sci. U.S.A. 121, e2407398121 (2024).","DOI":"10.1073\/pnas.2407398121"},{"key":"2026091118350951000_2026.09.04.749274v1.26","doi-asserted-by":"crossref","first-page":"3986","DOI":"10.3390\/ijms24043986","article-title":"What\u2019s on the other side of the gate: a structural perspective on DNA gate opening of type IA and IIA DNA topoisomerases","volume":"24","year":"2023","journal-title":"Int. J. Mol. Sci"},{"key":"2026091118350951000_2026.09.04.749274v1.27","doi-asserted-by":"crossref","first-page":"2438","DOI":"10.3390\/ijms18112438","article-title":"Non-catalytic roles of the topoisomerase II\u03b1 C-terminal domain","volume":"18","year":"2017","journal-title":"Int. J. Mol. Sci"},{"key":"2026091118350951000_2026.09.04.749274v1.28","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0900574106"},{"key":"2026091118350951000_2026.09.04.749274v1.29","doi-asserted-by":"crossref","first-page":"e67021","DOI":"10.7554\/eLife.67021","article-title":"Topoisomerase VI is a chirally-selective, preferential DNA decatenase","volume":"11","year":"2022","journal-title":"eLife"},{"key":"2026091118350951000_2026.09.04.749274v1.30","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2004.05.034"},{"key":"2026091118350951000_2026.09.04.749274v1.31","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2006.06.005"},{"key":"2026091118350951000_2026.09.04.749274v1.32","doi-asserted-by":"publisher","DOI":"10.1529\/biophysj.105.076778"},{"key":"2026091118350951000_2026.09.04.749274v1.33","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkp041"},{"key":"2026091118350951000_2026.09.04.749274v1.34","doi-asserted-by":"crossref","first-page":"031902","DOI":"10.1103\/PhysRevE.81.031902","article-title":"Local site preference rationalizes disentangling by DNA topoisomerases","volume":"81","year":"2010","journal-title":"Phys. Rev. E: Stat., Nonlinear, Soft Matter Phys"},{"key":"2026091118350951000_2026.09.04.749274v1.35","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2010.05.007"},{"key":"2026091118350951000_2026.09.04.749274v1.36","first-page":"354103","article-title":"Consistent rationalization of type-2 topoisomerases\u2019 unknotting decatenating, supercoil-relaxing actions and their scaling relation","volume":"27","year":"2015","journal-title":"J. Phys.: Condens. Matter"},{"key":"2026091118350951000_2026.09.04.749274v1.37","doi-asserted-by":"crossref","first-page":"e31724","DOI":"10.7554\/eLife.31724","article-title":"Topoisomerase VI senses and exploits both DNA crossings and bends to facilitate strand passage","volume":"7","year":"2018","journal-title":"eLife"},{"key":"2026091118350951000_2026.09.04.749274v1.38","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkt136"},{"key":"2026091118350951000_2026.09.04.749274v1.39","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1631550100"},{"key":"2026091118350951000_2026.09.04.749274v1.40","doi-asserted-by":"publisher","DOI":"10.1529\/biophysj.104.056945"},{"key":"2026091118350951000_2026.09.04.749274v1.41","doi-asserted-by":"publisher","DOI":"10.1529\/biophysj.105.060202"},{"key":"2026091118350951000_2026.09.04.749274v1.42","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2958.2004.04047.x"},{"key":"2026091118350951000_2026.09.04.749274v1.43","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(90)90172-B"},{"key":"2026091118350951000_2026.09.04.749274v1.44","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.271.35.21529"},{"key":"2026091118350951000_2026.09.04.749274v1.45","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1133178100"},{"key":"2026091118350951000_2026.09.04.749274v1.46","doi-asserted-by":"publisher","DOI":"10.1101\/gad.838900"},{"key":"2026091118350951000_2026.09.04.749274v1.47","doi-asserted-by":"publisher","DOI":"10.1016\/j.bpj.2009.02.032"},{"key":"2026091118350951000_2026.09.04.749274v1.48","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2005.06.029"},{"key":"2026091118350951000_2026.09.04.749274v1.49","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-61779-282-3_15"},{"key":"2026091118350951000_2026.09.04.749274v1.50","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-61779-261-8_15"},{"key":"2026091118350951000_2026.09.04.749274v1.51","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1206480109"},{"key":"2026091118350951000_2026.09.04.749274v1.52","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.R109.092437"},{"key":"2026091118350951000_2026.09.04.749274v1.53","doi-asserted-by":"crossref","unstructured":"M. Pereira , C. Brackley , J. S. Lintuvuori , D. Marenduzzo , E. Orlandini , Entropic elasticity and dynamics of the bacterial chromosome: A simulation study. J. Chem. Phys. 147 (2017).","DOI":"10.1063\/1.4995992"},{"key":"2026091118350951000_2026.09.04.749274v1.54","doi-asserted-by":"crossref","unstructured":"C. A. Brackley , S. Taylor , A. Papantonis , P. R. Cook , D. Marenduzzo , Nonspecific bridging-induced attraction drives clustering of DNA-binding proteins and genome organization. Proc. Natl. Acad. Sci. U.S.A. 110, E3605-E3611 (2013).","DOI":"10.1073\/pnas.1302950110"},{"key":"2026091118350951000_2026.09.04.749274v1.55","doi-asserted-by":"publisher","DOI":"10.1016\/j.bpj.2015.10.027"},{"key":"2026091118350951000_2026.09.04.749274v1.56","doi-asserted-by":"crossref","first-page":"1877","DOI":"10.1039\/C4SM02198C","article-title":"A polymer in a crowded and confined space: effects of crowder size and poly-dispersity","volume":"11","year":"2015","journal-title":"Soft Matter"},{"key":"2026091118350951000_2026.09.04.749274v1.57","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0605305103"},{"key":"2026091118350951000_2026.09.04.749274v1.58","doi-asserted-by":"publisher","DOI":"10.1006\/jcph.1995.1039"},{"key":"2026091118350951000_2026.09.04.749274v1.59","doi-asserted-by":"crossref","first-page":"015012","DOI":"10.1088\/0965-0393\/18\/1\/015012","article-title":"Visualization and analysis of atomistic simulation data with OVITO\u2013the Open Visualization Tool","volume":"18","year":"2010","journal-title":"Modell. Simul. Mater. Sci. Eng"},{"key":"2026091118350951000_2026.09.04.749274v1.60","doi-asserted-by":"crossref","first-page":"e67021","DOI":"10.7554\/eLife.67021","article-title":"Topoisomerase VI is a chirally-selective, preferential DNA decatenase","volume":"11","year":"2022","journal-title":"eLife"},{"key":"2026091118350951000_2026.09.04.749274v1.61","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0900574106"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.09.04.749274","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,12]],"date-time":"2026-09-12T01:35:19Z","timestamp":1789176919000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.09.04.749274"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,8]]},"references-count":61,"URL":"https:\/\/doi.org\/10.64898\/2026.09.04.749274","relation":{},"subject":[],"published":{"date-parts":[[2026,9,8]]},"subtype":"preprint"}}