Benzo[c]phenanthrene Adducts and Nogalamycin Inhibit DNA Transesterification by Vaccinia Topoisomerase

Vaccinia DNA topoisomerase forms a covalent DNA-(3′-phosphotyrosyl)-enzyme intermediate at a specific target site 5′-C+5C+4C+3T+2T+1p↓N-1 in duplex DNA. Here we study the effects of position-specific DNA intercalators on the rate and extent of single-turnover DNA transesterification. Chiral C-1 R and S trans-opened 3,4-diol 1,2-epoxide adducts of benzo[c]phenanthrene (BcPh) were introduced at single N2-deoxyguanosine and N6-deoxyadenosine positions within the 3′-G+5G+4G+3A+2A+1T-1A-2 sequence of the nonscissile DNA strand. Transesterification was unaffected by BcPh intercalation between the +6 and +5 base pairs, slowed 4-fold by intercalation between the +5 and +4 base pairs, and virtually abolished by BcPh intercalation between the +4 and +3 base pairs and the +3 and +2 base pairs. Intercalation between the +2 and +1 base pairs by the +2R BcPh dA adduct abolished transesterification, whereas the overlapping +1S BcPh dA adduct slowed the rate of transesterification by a factor of 2700, with little effect upon the extent of the reaction. Intercalation at the scissile phosphodiester (between the +1 and -1 base pairs) slowed transesterification by a factor of 450. BcPh intercalation between the -1 and -2 base pairs slowed cleavage by two orders of magnitude, but intercalation between the -2 and -3 base pairs had little effect. The anthracycline drug nogalamycin, a non-covalent intercalator with preference for 5′-TG dinucleotides, inhibited the single-turnover DNA cleavage reaction of vaccinia topoisomerase with an IC50 of 0.7 μm. Nogalamycin was most effective when the drug was pre-incubated with DNA and when the cleavage target site was 5′-CCCTT↓G instead of 5′-CCCTT↓A. These findings demarcate upstream and downstream boundaries of the functional interface of vaccinia topoisomerase with its DNA target site.