Stimulation of topoisomerase II-mediated DNA damage via a mechanism involving protein thiolation

The breakage/reunion reaction of DNA topoisomerase II (TOP2) can be interru pted by DNA intercalators (e.g., doxorubicin), enzyme binders (e.g., etopos ide), or DNA lesions (e.g., abasic sites) to produce TOP2-mediated DNA dama ge. Here, we demonstrate that thiol alkylation of TOP2 can also produce TOP 2-mediated DNA damage. This conclusion is supported by the following observ ations using purified TOP2: (1) Thiol-reactive quinones were shown to induc e TOP2-mediated DNA cleavage, (2) Thiol-reactive compounds such as N-ethylm aleimide (NEM), disulfiram, and organic disulfides [e.g., 2,2 ' -dithiobis( 5-nitropyridine)] were also shown to induce TOP2-mediated DNA cleavage with similar reaction characteristics as thiol-reactive quinones. (3) TOP2-medi ated DNA cleavage induced by thiol-reactive quinones was completely abolish ed using mutant yeast TOP2 with all cysteine residues replaced with alanine (cysteineless TOP2). These results suggest the possibility that cellular D NA damage could occur indirectly through thiolation of a nuclear protein, T OP2. The implications of this reaction in carcinogenesis and apoptotic cell death are discussed.