CAMPTOTHECINS INHIBIT THE UTILIZATION OF HYDROGEN-PEROXIDE IN THE LIGATION STEP OF TOPOISOMERASE-I CATALYSIS

The antitumor compounds camptothecin and its derivatives topotecan and irinotecan stabilize topoisomerase I cleavage complexes by inhibiting the religation reaction of the enzyme. Previous studies, using radiol abeled camptothecin or affinity labeling reagents structurally related to camptothecin, suggest that the agent binds at the topoisomerase I- DNA interface of the cleavage complexes, interacting with both the cov alently bound enzyme and with the +1 base. In this study, we have inve stigated the molecular mechanism of camptothecin action further by tak ing advantage of the ability of topoisomerase I to couple non-DNA nucl eophiles to the cleaved strand of the covalent enzyme-DNA complexes. T his reaction of topoisomerase I was originally observed at moderate ba sic pH where active cleavage complexes mediate hydrolysis or alcoholys is by accepting water or polyhydric alcohol compounds as substitutes f or a 5'-OH DNA end in the ligation step. Here, we report that a H2O2-d erived nucleophile, presumably, the peroxide anion, facilitates the re lease of topoisomerase I from the cleavage complexes at neutral pH, an d we present evidence showing that this reaction is mechanistically an alogous to DNA ligation. We find that camptothecin, topotecan, and SN- 38 (the active metabolite of irinotecan) inhibit H2O2 ligation mediate d by cleavage complexes not containing DNA downstream of the cleavage site, indicating that drug interaction with DNA 3' to the covalently b ound enzyme is not strictly required for the inhibition, although the presence of double-stranded DNA in this region enhances the drug effec t. The results suggest that camptothecins prevent ligation by blocking the active site of the covalently bound enzyme.