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

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 gamma-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 gamma-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 gamma-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 gamma-radiation-induced 'ladder'. The 'ladder' was not observed after repair incubation, UV-irradiation or gamma-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 gamma-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.