Nucleolytic degradation of 3'-ending overhangs is essential for DNA-end resection in RecA-loading deficient recB mutants of Escherichia coli.

Degradation of a 5'-ending strand is the hallmark of the universal process of DNA double strand break (DSB) resection, which results in creation of the central recombination intermediate, a 3'-ending overhang. Here we show that in Escherichia coli recB1080/recB1067 mutants, which are devoid of RecBCD's nuclease and RecA loading activities, degradation of the unwound 3' tail is as essential as is degradation of its 5'-ending complement. Namely, a synergistic action of ExoI, ExoVII, SbcCD and ExoX single-strand specific exonucleases (ssExos) of 3'-5' polarity was essential for preserving cell viability, DNA repair and homologous recombination in the recB1080/recB1067 mutants, to the same extent as the redundant action of 5'-tail trimming ssExos RecJ and ExoVII. recB1080 derivatives lacking 3'-5' ssExos also showed a strong induction of the SOS response and greatly increased SOS-dependent mutagenesis. Furthermore, we show that ExoI and ExoVII ssExos act synergistically in suppressing illegitimate recombination in the recB1080 mutant but not in a wt strain, while working in concert with the RecQ helicase. Remarkably, 3'-5' ssExos show synergism with RecQ helicase in the recB1080 mutant in all the assays tested. The effect of inactivation of 3'-5' ssExos in the recB1080/recB1067 mutants was much stronger than in wt, recD, and recB strains. These results demonstrate that the presence of a long, reactive 3' overhang can be as toxic for a cell as its complete absence, i.e. it may prevent DSB repair. Our results indicate that coupling of helicase and RecA-loading activity during dsDNA-end resection is crucial in avoiding the deleterious effects of a long and stabile 3' tail in E. coli.

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