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Papers In Press, published online ahead of print March 23, 2004
Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021
Corresponding Author: s-shuman{at}ski.mskcc.org
Vaccinia DNA topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a specific target site 5'-C+5C+4C+3T+2T+1pN-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 by 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 on 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 noncovalent 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 preincubated with DNA and when the cleavage target site was 5’-CCCTTG instead of 5’-CCCTTA. These findings demarcate upstream and downstream boundaries of the functional interface of vaccinia topoisomerase with its DNA target site.
J. Biol. Chem, 10.1074/jbc.M401203200
Submitted on February 3, 2004
Revised on March 22, 2004
Accepted on March 23, 2004
Benzo[c]phenanthrene adducts and nogalamycin Inhibit DNA transesterification by vaccinia topoisomerase
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