Nucleic Acids Research, Vol 24, Issue 4 640-647, Copyright © 1996 by Oxford University Press
S Takamatsu, R Kato and S Kuramitsu
The mutS gene, implicated in DNA mismatch repair, was cloned from an
extremely thermophilic bacterium, Thermus thermophilus HB8. Its nucleotide
sequence encoded a 819-amino acid protein with a molecular mass of 91.4
kDa. Its predicted amino acid sequence showed 56 and 39% homology with
Escherichia coli MutS and human hMsh2 proteins, respectively. The
T.thermophilus mutS gene complemented the hypermutability of the E.coli
mutS mutant, suggesting that T.thermophilus MutS protein was active in
E.coli and could interact with E.coli MutL and/or MutH proteins. The
T.thermophilus mutS gene product was overproduced in E.coli and then
purified to homogeneity. Its molecular mass was estimated to be 91 kDa by
SDS-PAGE but approx. 330 kDa by size-exclusion chromatography, suggesting
that T.thermophilus MutS protein was a tetramer in its native state.
Circular dichroic measurements indicated that this protein had an alpha-
helical content of approx. 50%, and that it was stable between pH 1.5 and
12 at 25 degree C and was stable up to 80 degree C at neutral pH. Thermus
thermophilus MutS protein hydrolyzed ATP to ADP and Pi, and its activity
was maximal at 80 degrees C. The kinetic parameters of the ATPase activity
at 65 degrees C were Km = 130 microM and Kcat = 0.11 s(- 1). Thermus
thermophilus MutS protein bound specifically with G-T mismatched DNA even
at 60 degrees C.
ARTICLES
Mismatch DNA recognition protein from an extremely thermophilic bacterium, Thermus thermophilus HB8
Department of Biology, Faculty of Science, Osaka University, Toyonaka, Japan.
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