ESCHERICHIA-COLI MUTY PROTEIN HAS A GUANINE-DNA GLYCOSYLASE THAT ACTSON 7,8-DIHYDRO-8-OXOGUANINE-GUANINE MISPAIR TO PREVENT SPONTANEOUS G-C-]C-G TRANSVERSIONS

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. Es cherichia coli must have several pathways to repair such mismatches an d DNA modifications. In this study, we attempted to identify mutator l oci leading to G:C-->C:G transversions in E.coli. The strain CC103 car rying a specific mutation in lacZ was mutagenized by random miniTn10 i nsertion mutagenesis, In this strain, only the G:C-->C:G change can re vert the glutamic acid at codon 461, which is essential for sufficient beta-galactosidase activity to allow growth on lactose, Mutator strai ns were detected as colonies with significantly increased rates of pap illae formation on glucose minimal plates containing P-Gal and X-Gal. We screened similar to 40 000 colonies and selected several mutator st rains. The strain GC39 showed the highest mutation rate to Lac(+). The gene responsible for the mutator phenotypes, mut39, was mapped at aro und 67 min on the E.coli chromosome. The sequencing of the miniTn10-fl anking DNA region revealed that the mut39 was identical to the mutY ge ne of E.coli. The plasmid carrying the mutY(+) gene reduced spontaneou s G:C-->T:A and G:C-->C:G mutations in both mutY and mut39 strains. pu rified MutY protein bound to the oligonucleotides containing 7,8-dihyd ro-8-oxo-guanine (8oxoG):G and 8-oxoG:A, Furthermore, we found that th e 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 re moving guanine from the 8-oxoG:G mispair in E.coli.