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(Received for publication, September 9, 1996)
From the Molecular Biology Program, Sloan-Kettering Institute, New
York, New York 10021
Vaccinia topoisomerase catalyzes DNA cleavage and
rejoining via transesterification to pentapyrimidine recognition site
5
Volume 272, Number 7,
Issue of February 14, 1997
pp. 3891-3896
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
-(C/T)CCTT
in duplex DNA. The proposed reaction mechanism involves
general-base catalysis of the attack by active site nucleophile Tyr-274
on the scissile phosphodiester and general-acid catalysis of the expulsion of the 5-deoxyribose oxygen on the leaving DNA strand. The
pKa values suggest histidine and cysteine side
chains as candidates for the roles of proton acceptor and donor,
respectively. To test this, we replaced each of the eight histidines
and two cysteines of the vaccinia topoisomerase with alanine. Single
mutants C100A and C211A and a double mutant C100A-C211A were fully
active in DNA relaxation, indicating that a cysteine is not the general acid. Only one histidine mutation, H265A, affected enzyme activity. The
rates of DNA relaxation, single-turnover strand cleavage, and
single-turnover religation by H265A were 2 orders of magnitude lower
than the wild-type rates. Yet the H265A mutation did not alter the
dependence of the cleavage rate on pH, indicating that His-265 is not
the general base. Replacing His-265 with glutamine or asparagine slowed
DNA relaxation and single-turnover cleavage to about one-third of the
wild-type rate. All three mutations, H265A, H265N, and H265Q, skewed
the cleavage-religation equilibrium in favor of the covalently bound
state. His-265 is strictly conserved in every member of the eukaryotic
type I topoisomerase family.
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