engleski

3'-terminal Overhangs Regulate DNA Double-Strand Break Processing in Escherichia coli

Although double-strand breaks (DSBs) are lethal DNA lesions, they are essential intermediates during meiosis. DSBs are mostly repaired by a universal and evolutionary conserved process of homologous recombination, which involves degradation of the broken molecule, a process known as DNA-end resection. To study DSB processing, we induced DSBs into the Escherichia coli chromosome by gamma irradiation and measured chromosomal degradation. We show that DNA degradation is regulated by RecA protein concentration and its rate of association with ssDNA. RecA decreased DNA degradation in wild- type, recB and recD strains, indicating that it is a general phenomenon in E. coli. On the other hand, DNA degradation was greatly reduced and unaffected by RecA in the recB1080 mutant (which produces long overhangs) and in a strain devoid of four exonucleases that degrade a 3’ tail (ssExos). 3’-5’ ssExos deficiency is epistatic to RecA deficiency concerning DNA degradation, suggesting that bound RecA is shielding 3’ tail from degradation by 3’-5’ ssExos. Since 3’-tail preservation is common to all these situations, we infer that RecA polymerization constitutes a subset of mechanisms for preserving the integrity of 3’ tails emanating from DSBs, along with 3’ tail’s massive length, or prevention of their degradation by inactivation of 3’-5’ ssExos. Thus, we conclude that 3’ overhangs are crucial in controlling the extent of DSB processing in E. coli. This study suggests a regulatory mechanism for DSB processing in E. coli, wherein 3’ tails impose a negative feedback loop on DSB processing reactions, specifically on helicase reloading onto dsDNA ends.

DNA degradation ; exonuclease activity ; RecA protein ; single-strand-specific exonucleases ; recB1080 mutant

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