Mutational analysis of 26 residues of vaccinia DNA topoisomerase identifies Ser-204 as important for DNA binding and cleavage.
Vaccinia DNA topoisomerase, a 314 amino acid type I enzyme, catalyzes the cleavage and rejoining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate formed at a specific target sequence, 5'-(C/T)CCTT downward arrow. To identify amino acids that participate in the DNA binding and transesterification steps, we introduced alanine substitutions at 18 positions within a centrally located 27 amino acid segment (181-RLYKPLLKLTDDSSPEEFLFNKLSERK-207) and at 8 positions near the N-terminus (1-MRALFYKDGK-10). All mutant proteins except two displayed wild-type activity in relaxing supercoiled DNA. F200A and S204A exhibited reduced rates of relaxation and were subjected to a kinetic analysis of the strand cleavage reaction under single-turnover and equilibrium conditions. The F200A and S204A mutations reduced the rate of single-turnover DNA cleavage by factors of 5 and 70, respectively. Both mutations shifted the cleavage-religation equilibrium in favor of the noncovalently bound state. The S204A mutation reduced the affinity of topoisomerase for CCCTT-containing DNA, but did not alter the site-specificity of DNA cleavage. Vaccinia residue Ser-204, which is conserved in all poxvirus topoisomerases, but not in the cellular homologues, may contribute to the unique cleavage site specificity of the poxvirus enzymes. Phe-200 is conserved in all members of the type IB topoisomerase family.
Wang
L K
LK
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021.
Wittschieben
J
J
Shuman
S
S
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004264:DNA Topoisomerases, Type I / Q000235:genetics* / Q000378:metabolism*; D004278:DNA, Superhelical / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012694:Serine / Q000235:genetics* / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*
D004278:DNA, Superhelical; D004279:DNA, Viral; D014764:Viral Proteins; D012965:Sodium Chloride; D012694:Serine; D004264:DNA Topoisomerases, Type I; D008274:Magnesium; D000409:Alanine