Critical role of RecA and RecF proteins in strand break rejoining and maintenance of fidelity of rejoining following γ-radiation-induced damage to pMTa4 DNA in E. coli

Abstract

Purpose: This study was undertaken to understand the roles of RecA and RecF proteins in strand break rejoining and maintenance of fidelity of the process following exposure of E. coli to γ-radiation in vivo.Materials and methods: A plasmid DNA construct, pMTa4, was transformed into isogenic repair proficient (wild) and deficient (recF and recA) E. coli strains and γ-irradiated up to 30 Gy in vivo. The plasmid DNA was isolated under repair non-permissive (R−)and permissive (R+) conditions and analyzed by gel electrophoresis for the yields of single strand breaks (SSB) and double strand breaks (DSB) and their repair. The clonogenic survival of the E. coli was also recorded. The effects of γ-irradiation on recA reconstituted with cell free extract of wild strain or ultra-violet (UV)-irradiation were also monitored.Results: None of the strains used in this investigation showed effects of radiation-induced oxidative base damage. The dose dependent increase in SSB and DSB on pMTa4 in wild and recF mutants in R− condition were abolished upon repair incubation. The recA mutant exhibited a disturbed yield of SSB and DSB along with formation of γ-radiation-induced ‘ladder’. The ‘ladder’ was not observed after repair incubation, UV-irradiation or γ-irradiation in presence of cell-free extract of wild strain. The survival of recA mutants was seriously compromised.Conclusions: Wild, recF and recA strains of E. coli could repair γ-irradiation-induced oxidative damage to base or nucleotide (NT) in vivo. In absence of either RecA or RecF proteins, efficiency of rejoining of strand went down; RecA proteins seemed more critical than RecF in this. High fidelity or correct rejoining of strand breaks, on the other hand, seemed to require simultaneous presence of both RecA and RecF proteins.