Nucleic Acids Research, Vol 26, Issue 2 490-496, Copyright © 1998 by Oxford University Press
J Wittschieben, BO Petersen and S Shuman
Vaccinia topoisomerase forms a covalent protein-DNA intermediate at 5'-
CCCTT downward arrow sites in duplex DNA. The T downward arrow nucleotide
is linked via a 3'-phosphodiester bond to Tyr-274 of the enzyme. Here, we
report that mutant enzymes containing glutamate, cysteine or histidine in
lieu of Tyr-274 catalyze endonucleolytic cleavage of a 60 bp duplex DNA at
the CCCTT downward arrow site to yield a 3' phosphate-terminated product.
The Cys-274 mutant forms trace levels of a covalent protein-DNA complex,
suggesting that the DNA cleavage reaction may proceed through a
cysteinyl-phosphate intermediate. However, the His-274 and Glu-274 mutants
evince no detectable accumulation of a covalent protein-DNA adduct. Glu-274
is the most active of the mutants tested. The pH dependence of the
endonuclease activity of Glu-274 (optimum pH = 6.5) is distinct from that
of the wild-type enzyme in hydrolysis of the covalent adduct (optimum pH =
9.5). At pH 6.5, the Glu-274 endonuclease reaction is slower by 5-6 orders
of magnitude than the rate of covalent adduct formation by the wild-type
topoisomerase, but is approximately 20 times faster than the rate of
hydrolysis by the wild-type covalent adduct. We discuss two potential
mechanisms to account for the apparent conversion of a topoisomerase into
an endonuclease.
ARTICLES
Replacement of the active site tyrosine of vaccinia DNA topoisomerase by glutamate, cysteine or histidine converts the enzyme into a site- specific endonuclease
Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA.
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