Effect of uvrC gene in mutation induction by 5-formyldeoxyuridine in Escherichia coli

Abstract

The oxidation of the methyl group of thymine by reactive oxygen species forms the mutagenic base residue 5-formyluracil (fU). fU can be removed from DNA in vitro by AlkA (3-methyl DNA glycosylase II) and the Fpg, Nth and Nei proteins of Escherichia coli, which initiate the base excision repair pathway. In addition, it has been reported that the mammalian nucleotide excision repair (NER) system exhibits activity for fU in DNA in vitro. We recently found that the E. coli NER protein UvrA is involved in 5-formyldeoxyuridine (fdU)-medi-ated mutagenesis in E. coli, which indicates a role in fU repair in vivo. In this study, spontaneous mutagenesis and mutagenesis caused by obtained of 0.1 mM fdU to the culture medium of E. coli deficient in the NER gene uvrC were investigated by scoring resistance to rifampicin. It was found that fdU does not affect the relative growth rate of uvrC cells significantly confirming the moderate toxicity of fdU. The mutation rate increased slightly by addition of fdU to the uvrC cells, as previously shown for uvrA cells. This contrasted with wild-type, alkA and uvrB cells which we found doubled their mutation rates by fdU supplementation. Thus the results show that mutagenesis caused by 0.1 mM fdU is highly dependent on the UvrC (and UvrA) protein, in contrast to AlkA or UvrB which only affect mutagenesis slightly. The almost lack of induction of AT  GC transitions in uvrC, which may be regarded as the “signature” mutation of fdU, underscore this characteristics. Compared to the results ob-tained on AlkA, this is surprising, because evidence of a similar important role for UvrC protein in fU repair has hitherto not been apparent.