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Vertebrate topoisomerase II\u03b1 (TOP2\u03b1) resolves catenanes to relieve topological stress and unlink daughter molecules during DNA replication. Topoisomerase III\u03b1 (TOP3\u03b1) can also resolve catenanes, but its direct role during DNA replication, substrate specificity, and relevant binding partners remain unclear. Here we show that TOP3\u03b1 becomes crucial when TOP2\u03b1 function is compromised. We find that in\n                  <jats:italic>Xenopus<\/jats:italic>\n                  egg extracts, TOP3\u03b1 promotes replication fork progression and daughter strand unlinking specifically during replication termination. TOP3\u03b1 can carry out this role independently of its binding partners RMI1-RMI2 and the BLM helicase by acting on lagging-strand single-stranded DNA (ssDNA). Strikingly, elevated lagging-strand ssDNA drives formation of intramolecular ssDNA intertwines, which are ordinarily resolved by TOP3\u03b1. Thus, TOP3\u03b1 resolves intermolecular linkages to promote fork progression during termination and resolves intramolecular linkages that arise when high levels of ssDNA are present during DNA replication.\n                <\/jats:p>","DOI":"10.64898\/2026.06.30.735647","type":"posted-content","created":{"date-parts":[[2026,7,1]],"date-time":"2026-07-01T16:10:14Z","timestamp":1782922214000},"source":"Crossref","is-referenced-by-count":0,"title":["Topoisomerase III\u03b1 resolves inter- and intra-molecular intertwines during DNA replication"],"prefix":"10.64898","author":[{"given":"Sabrina X","family":"Van Ravenstein","sequence":"first","affiliation":[{"name":"Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lillian V","family":"Campos","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gabor M","family":"Harami","sequence":"additional","affiliation":[{"name":"Laboratory of Single Molecule Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Darren R","family":"Heintzman","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Steven N","family":"Dahmen","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"William G","family":"Dunphy","sequence":"additional","affiliation":[{"name":"Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"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, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4153-3818","authenticated-orcid":false,"given":"James M.","family":"Dewar","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026070321100992000_2026.06.30.735647v1.1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.biochem.70.1.369"},{"key":"2026070321100992000_2026.06.30.735647v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/nrm.2016.111"},{"key":"2026070321100992000_2026.06.30.735647v1.3","doi-asserted-by":"crossref","unstructured":"A. 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