Immunodetection of human topoisomerase I-DNA covalent complexes

Anand G. Patel, Karen S. Flatten, Kevin L. Peterson, Thomas G. Beito, Paula A. Schneider, Angela L. Perkins, Daniel A. Harki, Scott H. Kaufmann

Research output: Contribution to journalArticlepeer-review

26 Scopus citations


A number of established and investigational anticancer drugs slow the religation step of DNA topoisomerase I (topo I). These agents induce cytotoxicity by stabilizing topo I-DNA covalent complexes, which in turn interact with advancing replication forks or transcription complexes to generate lethal lesions. Despite the importance of topo I-DNA covalent complexes, it has been difficult to detect these lesions within intact cells and tumors. Here, we report development of a monoclonal antibody that specifically recognizes covalent topo I-DNA complexes, but not free topo I or DNA, by immunoblotting, immunofluorescence or flow cytometry. Utilizing this antibody, we demonstrate readily detectable topo I-DNA covalent complexes after treatment with camptothecins, indenoisoquinolines and cisplatin but not nucleoside analogues. Topotecan-induced topo I-DNA complexes peak at 15-30 min after drug addition and then decrease, whereas indotecan-induced complexes persist for at least 4 h. Interestingly, simultaneous staining for covalent topo I-DNA complexes, phospho-H2AX and Rad51 suggests that topotecan-induced DNA double-strand breaks occur at sites distinct from stabilized topo I-DNA covalent complexes. These studies not only provide new insight into the action of topo I-directed agents, but also illustrate a strategy that can be applied to study additional topoisomerases and their inhibitors in vitro and in vivo.

Original languageEnglish (US)
Pages (from-to)2816-2826
Number of pages11
JournalNucleic acids research
Issue number6
StatePublished - Feb 24 2016

ASJC Scopus subject areas

  • Genetics


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