Mutations within a conserved region of vaccinia topoisomerase affect the DNA cleavage-religation equilibrium.
The segment of the vaccinia DNA topoisomerase from residues 143 to 167 (VGLLTLKNKHIEISPDEIVIKFVGK) is conserved in other members of the eukaryotic type I topoisomerase family. In order to gauge the function of this region, we performed a mutational analysis in which 23 of 25 positions were substituted by alanine. Several non-alanine mutations were also studied. Purified wild-type and mutant proteins were compared with respect to their activities in relaxing supercoiled DNA and in single-turnover strand cleavage. Lys167, an invariant residue, was judged essential for catalysis, insofar as alanine replacement resulted in a 100-fold decrement in specific activity. Alanine substitutions for invariant residues Gly144 and Gly166 were well-tolerated, but a G144R mutation inactivated the enzyme and G166R reduced activity by two orders of magnitude. More modest effects of other mutations were demonstrated by kinetic analysis of the single-turnover DNA cleavage and religation reactions and by studies of covalent adduct formation under equilibrium conditions. Mutations G144A and T147A elicited a shift in the cleavage-religation equilibrium toward the non-covalently bound state; this was caused by slowing of the forward cleavage reaction. Mutations F164A, G166A, G166R, K167A, and K167R produced opposite effects on reaction equilibrium, resulting in higher levels of covalent complex formation. We suggest that invariant residues F164, G166, and K167, constitute part of the active site of the enzyme.
Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA.
B O
BO
Petersen
J
J
Wittschieben
S
S
Shuman
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D004264:DNA Topoisomerases, Type I / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D013379:Substrate Specificity; D014616:Vaccinia virus / Q000201:enzymology / Q000235:genetics*
D004279:DNA, Viral; D004264:DNA Topoisomerases, Type I