Mutational analysis of vaccinia DNA topoisomerase defines amino acid residues essential for covalent catalysis.
The eukaryotic family of type I DNA topoisomerases includes the nuclear type I enzymes and the enzymes encoded by vaccinia and other poxviruses. The small size of the vaccinia topoisomerase (314 amino acids as compared to 765-972 amino acids for the cellular enzymes) makes it likely that this protein constitutes the minimal functional unit of a eukaryotic type I enzyme and provides an opportunity for a comprehensive structure-function analysis through mutagenesis. Two protein subregions were targeted for mutagenesis in the present study. The role of the Ser-Lys-X-X-Tyr sequence present at the active site of all family members was examined by replacing each conserved residue with alanine. Alanine substitution at the active site Tyr abrogated topoisomerase activity. In contrast, mutations at Ser-270 and Lys-271 had no effect on enzyme activity. The region of the vaccinia topoisomerase from amino acids 126-142 (MFFIRFGKMKYLKENET) is highly conserved and contains a residue, Gly-132, shown previously to be essential. Twenty-nine different mutations were generated in this region, with at least one substitution at each position. Point mutations were identified at three positions, Arg-130, Tyr-136, and Leu-137, which either abrogated or severely reduced DNA relaxation. The effects on activity could be attributed to a defect in formation of the covalent intermediate. Alterations of 13 other amino acids, including conserved residues, had little or no effect on topoisomerase activity.
Wittschieben
J
J
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021.
Shuman
S
S
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D017931:DNA Primers; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics
D017931:DNA Primers; D004268:DNA-Binding Proteins; D004247:DNA; D004264:DNA Topoisomerases, Type I; D000409:Alanine