Mutational analysis of vaccinia virus topoisomerase identifies residues involved in DNA binding.
Vaccinia DNA topoisomerase catalyzes the cleavage and re-joining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate formed at a specific target sequence, 5'-(C/T)CCTT downward arrow. The 314 aa protein consists of three protease-resistant structural domains demarcated by protease-sensitive interdomain segments referred to as the bridge and the hinge. The bridge is defined by trypsin-accessible sites at Arg80, Lys83 and Arg84. Photocrosslinking and proteolytic footprinting experiments suggest that residues near the interdomain bridge interact with DNA. To assess the contributions of specific amino acids to DNA binding and transesterification chemistry, we introduced alanine substitutions at 16 positions within a 24 aa segment from residues 63 to 86(DSKGRRQYFYGKMHVQNRNAKRDR). Assays of the rates of DNA relaxation under conditions optimal for the wild-type topoisomerase revealed significant mutational effects at six positions; Arg67, Tyr70, Tyr72, Arg80, Arg84 and Asp85. The mutated proteins displayed normal or near-normal rates of single-turnover transesterification to DNA. The effects of amino acid substitutions on DNA binding were evinced by inhibition of covalent adduct formation in the presence of salt and magnesium. The mutant enzymes also displayed diminished affinity for a subset of cleavage sites in pUC19 DNA. Tyr70 and Tyr72 were subjected to further analysis by replacement with Phe, His, Gln and Arg. At both positions, the aromatic moiety was important for DNA binding.
Sekiguchi
J
J
Molecular Biology Program, Sloan-Kettering Institute, 1275 York Avenue, New York, NY 10021, USA.
Shuman
S
S
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D004252:DNA Mutational Analysis; D004264:DNA Topoisomerases, Type I / Q000235:genetics* / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D014616:Vaccinia virus / Q000201:enzymology / Q000235:genetics*
D004279:DNA, Viral; D004264:DNA Topoisomerases, Type I