MUTATIONAL ANALYSIS OF VACCINIA VIRUS TOPOISOMERASE IDENTIFIES RESIDUES INVOLVED IN DNA-BINDING

Vaccinia DNA topoisomerase catalyzes the cleavage and re-joining of DN A strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate formed at a specific target sequence, 5'-(CTT)CCTT down arrow. The 314 aa pr otein consists of three protease-resistant structural domains demarcat ed by protease-sensitive interdomain segments referred to as the bridg e 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 interac t with DNA. To assess the contributions of specific amino acids to DNA binding and transesterification chemistry, we introduced alanine subs titutions at 16 positions within a 24 aa segment from residues 63 to 8 6 (DSKGRRQYFYGKMHVQNRNAKRDR). Assays of the rates of DNA relaxation un der conditions optimal for the wild-type topoisomerase revealed signif icant 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 a mino acid substitutions on DNA binding were evinced by inhibition of c ovalent adduct formation in the presence of salt and magnesium. The mu tant enzymes also displayed diminished affinity for a subset of cleava ge sites in pUC19 DNA, Tyr70 and Tyr72 were subjected to further analy sis by replacement with Phe, His, Gin and Arg, At both positions, the aromatic moiety was important for DNA binding.