title: Benzo[c]phenanthrene adducts and nogalamycin inhibit DNA transesterification by vaccinia topoisomerase.
abstract: Vaccinia DNA topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a specific target site 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p downward arrow 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(+5)G(+4)G(+3)A(+2)A(+1)T(-1)A(-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 microM. 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.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
authors.forename: Lyudmila
authors.initials: L
authors.lastname: Yakovleva
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Handy
authors.forename: Jane M
authors.initials: JM
authors.lastname: Sayer
authors.forename: Michael
authors.initials: M
authors.lastname: Pirrung
authors.forename: Donald M
authors.initials: DM
authors.lastname: Jerina
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D018736:DNA Adducts / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007364:Intercalating Agents / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009621:Nogalamycin / Q000378:metabolism; D009690:Nucleic Acid Conformation; D010616:Phenanthrenes / Q000378:metabolism; D014616:Vaccinia virus / Q000201:enzymology*
chemical_list: D018736:DNA Adducts; D007364:Intercalating Agents; D010616:Phenanthrenes; D004264:DNA Topoisomerases, Type I; C002615:benzo(c)phenanthrene; D009621:Nogalamycin