RecBCD- RecFOR-independent pathway of homologous recombination in Escherichia coli

Abstract The RecA protein is a key bacterial recombination enzyme that catalyzes pairing and strand exchange between homologous DNA duplexes. In Escherichia coli, RecA protein assembly on DNA is mediated either by the RecBCD or RecFOR protein complexes. Correspondingly, two recombination pathways, RecBCD and RecF (or RecFOR), are distinguished in E. coli . Inactivation of both pathways in recB(CD) recF(OR) mutants results in severe recombination deficiency. Here we describe a novel, RecBCD- RecFOR-independent (RecBFI) recombination pathway that is active in Δ recBCD sbcB15 sbcC(D) Δ recF(OR) mutants of E. coli . In transductional crosses, these mutants show only four-fold decrease of recombination frequency relative to the wild-type strain. At the same time they recombine 40- to 90-fold better than their sbcB + sbcC + and Δ sbcB sbcC counterparts. The RecBFI pathway strongly depends on recA , recJ and recQ gene functions, and moderately depends on recG and lexA functions. Inactivation of dinI , helD, recX , recN , radA , ruvABC and uvrD genes has a slight effect on RecBFI recombination. After exposure to UV and gamma irradiation, the Δ recBCD sbcB15 sbcC Δ recF mutants show moderately increased DNA repair proficiency relative to their sbcB + sbcC + and Δ sbcB sbcC counterparts. However, introduction of recA730 allele (encoding RecA protein with enhanced DNA binding properties) completely restores repair proficiency to Δ recBCD sbcB15 sbcC Δ recF mutants, but not to their sbcB + sbcC + and Δ sbcB sbcC derivatives. Fluorescence microscopy with UV-irradiated recA-gfp fusion mutants suggests that the kinetics of RecA filament formation might be slowed down in the RecBFI pathway. Inactivation of 3’-5’ exonucleases ExoVII, ExoIX and ExoX cannot activate the RecBFI pathway in Δ recBCD Δ sbcB sbcC Δ recF mutants. Taken together, our results show that the product of the sbcB15 allele is crucial for RecBFI pathway. Besides protecting 3’ overhangs, SbcB15 protein might play an additional, more active role in formation of the RecA filament.