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Here, we show that spontaneous or replication stress-induced chromatin bridges exhibit \u201cknots\u201d of catenated and overtwisted DNA next to the midbody. Topoisomerase II\u03b1 (Top2\u03b1) forms abortive Top2\u2013DNA cleavage complexes (Top2ccs) on DNA knots; furthermore, impaired Top2\u03b1\u2013DNA cleavage activity correlates with chromatin bridge breakage in cytokinesis. Proteasomal degradation of Top2ccs is required for Rad17 localization to Top2-generated double-strand DNA ends on DNA knots; in turn, Rad17 promotes local recruitment of the MRN complex and downstream ATM-Chk2-INCENP signaling to delay abscission and prevent chromatin breakage. In contrast, dicentric chromosomes that do not exhibit knotted DNA fail to activate the abscission checkpoint in human cells. These findings are the first to describe a mechanism by which the abscission checkpoint detects chromatin bridges, through generation of abortive Top2ccs on DNA knots, to preserve genome integrity.<\/jats:p>","DOI":"10.1083\/jcb.202303123","type":"journal-article","created":{"date-parts":[[2023,8,11]],"date-time":"2023-08-11T02:21:24Z","timestamp":1691720484000},"update-policy":"https:\/\/doi.org\/10.1083\/jcb.crossmarkpolicy","source":"Crossref","is-referenced-by-count":19,"title":["The abscission checkpoint senses chromatin bridges through Top2\u03b1 recruitment to DNA knots"],"prefix":"10.1083","volume":"222","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7693-3531","authenticated-orcid":true,"given":"Eleni","family":"Petsalaki","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00dr28g20","id-type":"ROR","asserted-by":"publisher"}],"name":"University of Crete 1 Department of Biology, , Heraklion, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-2066-2872","authenticated-orcid":true,"given":"Sofia","family":"Balafouti","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00dr28g20","id-type":"ROR","asserted-by":"publisher"}],"name":"University of Crete 1 Department of Biology, , Heraklion, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-4561-9714","authenticated-orcid":true,"given":"Athina A.","family":"Kyriazi","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00dr28g20","id-type":"ROR","asserted-by":"publisher"}],"name":"University of Crete 1 Department of Biology, , Heraklion, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8935-2106","authenticated-orcid":true,"given":"George","family":"Zachos","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00dr28g20","id-type":"ROR","asserted-by":"publisher"}],"name":"University of Crete 1 Department of Biology, , Heraklion, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"291","published-online":{"date-parts":[[2023,8,28]]},"reference":[{"key":"2023082914065465400_bib1","doi-asserted-by":"publisher","first-page":"2388","DOI":"10.1091\/mbc.e03-08-0558","article-title":"Human topoisomerase IIalpha: Targeting to subchromosomal sites of activity during interphase and mitosis","volume":"15","author":"Agostinho","year":"2004","journal-title":"Mol. 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