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Despite being a validated drug target and indispensable to the pathogen, only a limited repertoire of inhibitors targeting the enzyme have been identified. We employed monoclonal antibodies (mAbs) to address this shortfall. From the pool of a large number of mAbs, we describe an inhibitory mAb specific to mycobacterial TopoI with a distinct mechanism of action. Among the various steps of the TopoI reaction cycle, the mAb does not interfere with DNA binding but impedes DNA cleavage. It does not alter the religation activity of TopoI; however, it\u00a0inhibits its strand passage activity. Probing with the mAb, we show the precise step at which the topology of the DNA is changed during DNA relaxation reaction. Surprisingly, instead of the initial strand scission action of the enzyme, the subsequent strand passage followed by the second transesterification entails the alteration in DNA topology. With their selective and specific inhibitory properties, the mAb and its derived single-chain variable fragment (ScFv) would serve to probe the structure of mycobacterial TopoI and as a starting point in designing peptide inhibitors with therapeutic potential to combat the rampant drug-resistant M. tuberculosis.<\/jats:p>","DOI":"10.1093\/nar\/gkaf929","type":"journal-article","created":{"date-parts":[[2025,9,6]],"date-time":"2025-09-06T11:44:33Z","timestamp":1757159073000},"source":"Crossref","is-referenced-by-count":0,"title":["Inhibition of DNA cleavage and strand passage activities of\n                    <i>Mycobacterium tuberculosis<\/i>\n                    topoisomerase I"],"prefix":"10.1093","volume":"53","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-7766-2355","authenticated-orcid":false,"given":"Iqball","family":"Faheem","sequence":"first","affiliation":[{"name":"Indian Institute of Science Department of Microbiology and Cell Biology, , C.V. Raman Avenue , Bangalore , 560012 ,","place":["India"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2884-4654","authenticated-orcid":false,"given":"Majety Naga","family":"Leelaram","sequence":"additional","affiliation":[{"name":"Indian Institute of Science Department of Microbiology and Cell Biology, , C.V. Raman Avenue , Bangalore , 560012 ,","place":["India"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6728-5333","authenticated-orcid":false,"given":"Immadi Siva","family":"Ratnakar","sequence":"additional","affiliation":[{"name":"Indian Institute of Science Department of Microbiology and Cell Biology, , C.V. Raman Avenue , Bangalore , 560012 ,","place":["India"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rinkee","family":"Verma","sequence":"additional","affiliation":[{"name":"Indian Institute of Science Department of Microbiology and Cell Biology, , C.V. 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