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

Ivanković, Siniša; Vujaklija, Dušica; Đermić, Damir (2017) Nucleolytic degradation of 3′-ending overhangs is essential for DNA-end resection in RecA-loading deficient recB mutants of Escherichia coli. DNA repair, 57 . pp. 56-65. ISSN 1568-7864

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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.

Item Type: Article
Uncontrolled Keywords: recB1080 ; RecQ ; Single-strand specific DNA exonucleases ; DNA-end resection ; DSB repair
Subjects: NATURAL SCIENCES > Biology
NATURAL SCIENCES > Biology > Biochemistry and Molecular Biology
NATURAL SCIENCES > Biology > Genetics, Evolution and Phylogenetics
Divisions: Division of Molecular Biology
Division of Molecular Medicine
Project titleProject leaderProject codeProject type
Rekombinacija, popravak DNA i očuvanje integriteta genoma: novi puteviZahradka, DavorIP-2013-11-2978HRZZ
Depositing User: Damir Đermić
Date Deposited: 10 Nov 2022 14:16
DOI: 10.1016/j.dnarep.2017.06.024

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