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Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2026,9,8]]},"abstract":"<jats:p>\n                    DNA topoisomerase II (TOP2) generates transient DNA double-strand breaks that are trapped as TOP2-DNA covalent complexes (TOP2cc) by antibiotic and chemotherapy drugs. Here, we characterize tools for study of cellular responses to TOP2cc, exploiting a\n                    <jats:italic toggle=\"yes\">Saccharomyces cerevisiae<\/jats:italic>\n                    <jats:italic toggle=\"yes\">TOP2<\/jats:italic>\n                    mutant (\n                    <jats:italic toggle=\"yes\">TOP2-F1025Y,R1128G<\/jats:italic>\n                    ) that generates spontaneous and inhibitor-induced covalent complexes at elevated frequencies. This Top2-hc (for \u201chypercleavage\u201d) mutant protein inhibits yeast cell growth when expressed alone or with endogenous Top2, and growth defects are exacerbated in DNA-repair-deficient genetic backgrounds and\/or in the presence of low doses of the Top2 poison mAMSA. We generated analogous mutations in human and mouse TOP2A and TOP2B that gave increased TOP2cc, hypersensitization to topoisomerase poisons, increased DNA damage, and decreased cell survival in cultured cells. We further established knock-in mouse models with inducible, tissue-specific expression of each TOP2-hc isoform, demonstrating overt organismal toxicity and cellular markers of DNA damage responses. To illustrate the potential of these genetic tools, we carried out proof-of-principle screens in yeast and cultured human cells for sensitivity to TOP2-hc. The yeast screen revealed strong requirements for homologous recombination, moderate roles for sister chromatid cohesion and kinetochore function, and dependencies on vesicle and vacuolar functions. The pilot shRNA screen in human cells revealed shared requirements for resistance to expression of either TOP2A-hc or TOP2B-hc as well as examples of isoform specificity. These findings establish hypercleavage mutant proteins as effective tools for studying topoisomerase isoform-specific DNA damage and offer a foundation for exploring TOP2cc toxicity and tolerance in vivo.\n                  <\/jats:p>","DOI":"10.1073\/pnas.2616452123","type":"journal-article","created":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T17:58:11Z","timestamp":1788285491000},"update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":0,"title":["A multispecies toolkit of\n                    <i>TOP2<\/i>\n                    hypercleavage mutants for studying topoisomerase II\u2013mediated DNA damage"],"prefix":"10.1073","volume":"123","author":[{"given":"David","family":"Ontoso","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02yrq0923","id-type":"ROR","asserted-by":"publisher"}],"name":"Molecular Biology Program, Memorial Sloan Kettering Cancer Center","place":["New York"]},{"id":[{"id":"https:\/\/ror.org\/02yrq0923","id-type":"ROR","asserted-by":"publisher"}],"name":"HHMI, Memorial Sloan Kettering Cancer Center","place":["New York"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Monika","family":"Mehta","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02yrq0923","id-type":"ROR","asserted-by":"publisher"}],"name":"Molecular Biology Program, Memorial Sloan Kettering Cancer Center","place":["New York"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aidin","family":"Shabro","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02yrq0923","id-type":"ROR","asserted-by":"publisher"}],"name":"Molecular Biology Program, Memorial Sloan Kettering Cancer Center","place":["New York"]},{"id":[{"id":"https:\/\/ror.org\/02yrq0923","id-type":"ROR","asserted-by":"publisher"}],"name":"HHMI, Memorial Sloan Kettering Cancer Center","place":["New York"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John","family":"Dittmar","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00hj8s172","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biological Sciences, Columbia University","place":["New York"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert J. D.","family":"Reid","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01esghr10","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Genetics and Development (in Systems Biology), Columbia University Irving Medical Center","place":["New York"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rodney","family":"Rothstein","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01esghr10","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Genetics and Development (in Systems Biology), Columbia University Irving Medical Center","place":["New York"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John L.","family":"Nitiss","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02mpq6x41","id-type":"ROR","asserted-by":"publisher"}],"name":"Pharmaceutical Sciences Department, Retzky College of Pharmacy, University of Illinois Chicago","place":["Rockford"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1283-6417","authenticated-orcid":false,"given":"Scott","family":"Keeney","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02yrq0923","id-type":"ROR","asserted-by":"publisher"}],"name":"Molecular Biology Program, Memorial Sloan Kettering Cancer Center","place":["New York"]},{"id":[{"id":"https:\/\/ror.org\/02yrq0923","id-type":"ROR","asserted-by":"publisher"}],"name":"HHMI, Memorial Sloan Kettering Cancer Center","place":["New York"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"341","published-online":{"date-parts":[[2026,9,1]]},"reference":[{"key":"e_1_3_4_1_2","doi-asserted-by":"publisher","DOI":"10.1038\/nrm.2016.111"},{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41580-022-00452-3"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.biochem.1c00240"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.dnarep.2024.103751"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.add4920"},{"key":"e_1_3_4_6_2","doi-asserted-by":"publisher","DOI":"10.1111\/gtc.70076"},{"key":"e_1_3_4_7_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4614-0323-4_18"},{"key":"e_1_3_4_8_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2020.05.040"},{"key":"e_1_3_4_9_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2302064120"},{"key":"e_1_3_4_10_2","doi-asserted-by":"publisher","DOI":"10.1021\/cb300648v"},{"key":"e_1_3_4_11_2","doi-asserted-by":"publisher","DOI":"10.3390\/cancers16040680"},{"key":"e_1_3_4_12_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0079202"},{"key":"e_1_3_4_13_2","doi-asserted-by":"publisher","DOI":"10.1093\/genetics\/133.4.799"},{"key":"e_1_3_4_14_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.272.19.12801"},{"key":"e_1_3_4_15_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0026-895X(24)12375-9"},{"key":"e_1_3_4_16_2","doi-asserted-by":"publisher","DOI":"10.31083\/j.fbl2703093"},{"key":"e_1_3_4_17_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0156439"},{"key":"e_1_3_4_18_2","doi-asserted-by":"publisher","DOI":"10.1101\/gr.109033.110"},{"key":"e_1_3_4_19_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2008721117"},{"key":"e_1_3_4_20_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M804058200"},{"key":"e_1_3_4_21_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4939-7459-7_20"},{"key":"e_1_3_4_22_2","first-page":"4467","article-title":"Amsacrine and etoposide hypersensitivity of yeast cells overexpressing DNA topoisomerase II","volume":"52","author":"Nitiss J. 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