RecBCD coordinates repair of two ends at a DNA double-strand break, preventing aberrant chromosome amplification

Abstract

DNA double-strand break (DSB) repair is critical for cell survival. A diverse range of organisms from bacteria to humans rely on homologous recombination for accurate DSB repair. This requires both coordinate action of the two ends of a DSB and stringent control of the resultant DNA replication to prevent unwarranted DNA amplification and aneuploidy. In Escherichia coli, RecBCD enzyme is responsible for the initial steps of homologous recombination. Previous work has revealed recD mutants to be nuclease defective but recombination proficient. Despite this proficiency, we show here that a recD null mutant is defective for the repair of a two-ended DSB and that this defect is associated with unregulated chromosome amplification and defective chromosome segregation. Our results demonstrate that RecBCD plays an important role in avoiding this amplification by coordinating the two recombining ends in a manner that prevents divergent replication forks progressing away from the DSB site.

Replication-transcription conflicts trigger extensive DNA degradation in Escherichia coli cells lacking RecBCD

“…This model not only struggles to explain the symmetrical degradation in our ΔrecB cells where the chromosome is linearized at +200 kb, but it also struggles to explain why over-replication of the termination area is observed in ΔrecD single mutants [45]. Since two-ended dsDNA breaks are not repaired efficiently in the absence of RecD [64], the absence of RecD should still result in at least some depletion of sequences at break sites. Instead, over-replication is observed in the termination area of ΔrecD cells [45].…”

A new role for Escherichia coli Dam DNA methylase in prevention of aberrant chromosomal replication

“…Different models have been put forth earlier for reverse restart-like mechanisms during Red-mediated recombination in phage λ (76) and in E. coli recG (24,29) or recD (77) mutants, any of which could apply in the dam mutant. The feature common to all of them, and which distinguishes them from normal replication restart, is that whereas in the latter there is one terminus-directed replisome that is assembled with the aid of PriABC and DnaT proteins following recombinational repair of a DSE, reverse restart results in a total of three replisomes, one directed towards oriC and two towards the terminus.…”

DNA targeting and interference by a bacterial Argonaute nuclease

“…4b). The size of the smDNA peak was smaller in a strain with a mutated I-SceI site, which is cleaved less efficiently 25 , demonstrating that smDNA production depends on the efficiency of DSB formation (Fig. 3b).…”

“…The link between CbAgo-bound smDNAs and RecBCD suggests that DSBs might serve as a source for smDNA processing. To directly test the role of DSBs in smDNA biogenesis, we analysed CbAgo-associated smDNAs in bacterial strains with engineered DSBs, induced by either expression of the I-SceI meganuclease, recognizing its respective site in the genome 25 , or by a long palindrome (pal) processed by the host SbcCD (Mre11-Rad50) complex 26 (both inserted in the lac locus) (Fig. 3a).…”