SITE-SPECIFIC INTERACTION OF VACCINIA VIRUS TOPOISOMERASE-I WITH BASEAND SUGAR MOIETIES IN DUPLEX DNA

Vaccinia DNA topoisomerase specifically binds and forms a covalent add uct at DNA sites containing a conserved sequence element 5'(C/T)CCTT d own in the scissile strand. The molecular interactions that contribute to recognition of the CCCTT motif in a synthetic DNA substrate have b een examined using modification interference, modification protection, and analog substitution techniques. We report that topoisomerase make s contact with guanine nucleotide bases of the pentamer motif compleme ntary strand (3'GGGAA) within the major groove of the DNA helix and th at alteration of the binding surface by chemical modification is delet erious to the interaction. Additional contacts are made with guanine r esidues located outside the pentamer element. The enzyme is unable to form a covalent adduct with synthetic RNA substrates. Analysis of the cleavage of DNA duplexes containing 2'OMe sugars suggests that the ina bility of the vaccinia topoisomerase to cleave either an RNA duplex or an RNA:DNA hybrid can be accounted for by the interfering effects of a 2' sugar substituent at two or more sites within the pentamer. Inter action with the sugar at the +2T nucleotide appears to be the most cri tical, as judged by the effects of single sugar substitutions.