Site-specific interaction of vaccinia virus topoisomerase I with base and sugar moieties in duplex DNA.
Vaccinia DNA topoisomerase specifically binds and forms a covalent adduct at DNA sites containing a conserved sequence element 5'(C/T)CCTT decreases in the scissile strand. The molecular interactions that contribute to recognition of the CCCTT motif in a synthetic DNA substrate have been examined using modification interference, modification protection, and analog substitution techniques. We report that topoisomerase makes contact with guanine nucleotide bases of the pentamer motif complementary strand (3'GGGAA) within the major groove of the DNA helix and that alteration of the binding surface by chemical modification is deleterious to the interaction. Additional contacts are made with guanine residues 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 inability 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. Interaction with the sugar at the +2T nucleotide appears to be the most critical, as judged by the effects of single sugar substitutions.
Shuman
S
S
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021.
Turner
J
J
D001483:Base Sequence; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D007288:Inosine; D008745:Methylation; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D009711:Nucleotides / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D013379:Substrate Specificity; D014498:Uracil; D014616:Vaccinia virus / Q000201:enzymology*
D009711:Nucleotides; D014498:Uracil; D007288:Inosine; D012313:RNA; D004247:DNA; D004264:DNA Topoisomerases, Type I