Evidence for involvement of UvrB in elicitation of ‘SIR’ phenotype by rpoB87-gyrA87 mutations in lexA3 mutant of Escherichia coli

Abstract

An unconventional DNA repair termed SIR (SOS Independent Repair), specific to mitomycin C (MMC) damage elicited by a combination of specific RifR (rpoB87) and NalR (gyrA87) mutations in SOS un-inducible strains of Escherichia coli was reported by Kumaresan and Jayaraman (1988). We report here that the rpoB87 mutation defines a C1565→T1565 transition changing S522→F522 and gyrA87 defines a G244→A244 transition changing D82→N82. The reconstructed lexA3 rpoB87 gyrA87 strain (DM49RN) exhibited resistance to MMC but not to UV as expected. When mutations in several genes implicated in SOS/NER were introduced into DM49RN strain, uvrB mutation alone decreased the MMC resistance and suppressed SIR phenotype. This was alleviated about two fold by a plasmid clone bearing the uvrB+ allele. Neither SulA activity as measured based on filamentation and sulA::gfp fluorescence analyses nor the transcript levels of sulA as seen based on RT-PCR analyses indicate a change in sulA expression in DM49RN strain. However, uvrB transcript levels are increased with or without MMC treatment in the same strain. While the presence of lexA3 allele in a plasmid clone was found to markedly decrease the MMC resistance of the DM49RN strain, the additional presence of uvrB+ allele in the same clone alleviated the suppression of MMC resistance by lexA3 allele to a considerable extent. These results indicate the increased expression of uvrB in the DM49RN strain is probably from the LexA dependent promoter of uvrB. The sequence analyses of various uvrB mutants including those isolated in this study using localized mutagenesis indicate the involvement of the nucleotide phosphate binding domain (ATPase domain) and the ATP binding domain and/or the DNA binding domain of the UvrB protein in the MMC repair in DM49RN. The possible involvement of UvrB protein in the MMC damage repair in DM49RN strain in relation to DNA repair is discussed.