Nucleic Acids Research, Vol 26, Issue 20 4669-4675, Copyright © 1998 by Oxford University Press
QM Zhang, N Ishikawa, T Nakahara and S Yonei
Low rates of spontaneous G:C-->C:G transversions would be achieved not
only by the correction of base mismatches during DNA replication but also
by the prevention and removal of oxidative base damage in DNA. Escherichia
coli must have several pathways to repair such mismatches and DNA
modifications. In this study, we attempted to identify mutator loci leading
to G:C-->C:G transversions in E.coli. The strain CC103 carrying a
specific mutation in lacZ was mutagenized by random miniTn 10 insertion
mutagenesis. In this strain, only the G:C-->C:G change can revert the
glutamic acid at codon 461, which is essential for sufficient
beta-galactosidase activity to allow growth on lactose. Mutator strains
were detected as colonies with significantly increased rates of papillae
formation on glucose minimal plates containing P-Gal and X-Gal. We screened
approximately 40 000 colonies and selected several mutator strains. The
strain GC39 showed the highest mutation rate to Lac+. The gene responsible
for the mutator phenotypes, mut39 , was mapped at around 67 min on the
E.coli chromosome. The sequencing of the miniTn 10 -flanking DNA region
revealed that the mut39 was identical to the mutY gene of E.coli. The
plasmid carrying the mutY + gene reduced spontaneous G:C-->T:A and
G:C-->C:G mutations in both mutY and mut39 strains. Purified MutY
protein bound to the oligonucleotides containing 7,8-dihydro-8-oxo-guanine
(8-oxoG):G and 8-oxoG:A. Furthermore, we found that the MutY protein had a
DNA glycosylase activity which removes unmodified guanine from the 8-oxoG:G
mispair. These results demonstrate that the MutY protein prevents the
generation of G:C-->C:G transversions by removing guanine from the
8-oxoG:G mispair in E.coli.
ARTICLES
Escherichia coli MutY protein has a guanine-DNA glycosylase that acts on 7,8-dihydro-8-oxoguanine:guanine mispair to prevent spontaneous G:C-- >C:G transversions
Laboratory of Radiation Biology, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.
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