Nucleic Acids Research, Vol 26, Issue 4 903-910, Copyright © 1998 by Oxford University Press
T Mikawa, R Kato, M Sugahara and S Kuramitsu
The mutM (fpg) gene, which encodes a DNA glycosylase that excises an
oxidatively damaged form of guanine, was cloned from an extremely
thermophilic bacterium, Thermus thermophilus HB8. Its nucleotide sequence
encoded a 266 amino acid protein with a molecular mass of approximately 30
kDa. Its predicted amino acid sequence showed 42% identity with the
Escherichia coli protein. The amino acid residues Cys, Asn, Gln and Met,
known to be chemically unstable at high temperatures, were decreased in
number in T.thermophilus MutM protein compared to those of the E.coli one,
whereas the number of Pro residues, considered to increase protein
stability, was increased. The T.thermophilus mutM gene complemented the
mutability of the E.coli mutM mutY double mutant, suggesting that T.
thermophilus MutM protein was active in E.coli. The T.thermophilus MutM
protein was overproduced in E.coli and then purified to homogeneity.
Size-exclusion chromatography indicated that T. thermophilus MutM protein
exists as a more compact monomer than the E.coli MutM protein in solution.
Circular dichroism measurements indicated that the alpha-helical content of
the protein was approximately 30%. Thermus thermophilus MutM protein was
stable up to 75 degrees C at neutral pH, and between pH 5 and 11 and in the
presence of up to 4 M urea at 25 degrees C. Denaturation analysis of
T.thermophilus MutM protein in the presence of urea suggested that the
protein had at least two domains, with estimated stabilities of 8.6 and
16.2 kcal/mol-1, respectively. Thermus thermophilus MutM protein showed
8-oxoguanine DNA glycosylase activity in vitro at both low and high
temperatures.
ARTICLES
Thermostable repair enzyme for oxidative DNA damage from extremely thermophilic bacterium, Thermus thermophilus HB8 [published erratum appears in Nucleic Acids Res 1998 Apr 1;26(7):following 1855]
Department of Biology, Graduate School of Science, Osaka University, 1- 1 Machikaneyama-cho, Toyonaka, Osaka 560, Japan.
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