Nucleic Acids Research, Vol 25, Issue 15 3001-3008, Copyright © 1997 by Oxford University Press
J Wittschieben and S Shuman
Vaccinia topoisomerase, a eukaryotic type IB enzyme, catalyzes relaxation
of supercoiled DNA by cleaving and rejoining DNA strands through a DNA-
(3'-phosphotyrosyl)-enzyme intermediate. We have performed a kinetic
analysis of mutational effects at four essential amino acids: Arg-130,
Gly-132, Tyr-136 and Lys-167. Arg-130, Gly-132 and Lys-167 are conserved in
all members of the type IB topoisomerase family. Tyr-136 is conserved in
all poxvirus topoisomerases. We show that Arg-130 and Lys-167 are required
for transesterification chemistry. Arg-130 enhances the rates of both
cleavage and religation by 10(5). Lys-167 enhances the cleavage and
religation reactions by 10(3) and 10(4), respectively. An instructive
distinction between these two essential residues is that Arg-130 cannot be
replaced by lysine, whereas substituting Lys-167 by arginine resulted in
partial restoration of function relative to the alanine mutant. We propose
that both basic residues interact directly with the scissile phosphate at
the topoisomerase active site. Mutations at positions Gly-132 and Tyr- 136
reduced the rate of strand cleavage by more than two orders of magnitude,
but elicited only mild effects on religation rate. Gly-132 and Tyr-136 are
suggested to facilitate a pre-cleavage activation step. The results of
comprehensive mutagenesis of the vaccinia topoisomerase illuminate
mechanistic and structural similarities to site-specific recombinases.
ARTICLES
Mechanism of DNA transesterification by vaccinia topoisomerase: catalytic contributions of essential residues Arg-130, Gly-132, Tyr-136 and Lys-167
Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA.
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