Dynamics of DNA Double-Strand Break Repair in Bacillus subtilis
Humberto Sanchez, Begoña Carrasco, Silvia Ayora and Juan C. Alonso
from Bacillus: Cellular and Molecular Biology Download flyer
All organisms have developed a variety of repair mechanisms, with recombination being the ultimate step for DNA repair and for promoting re-establishment of replication forks that are stalled or collapsed. This review summarises our current knowledge on the cellular response to DNA damage in Bacillus subtilis cells. Cytological approaches now confirm previous observations from genetic and biochemical analyses, which suggested that recombinational repair, and especially double-strand break repair, is choreographed by multi-protein complexes that are organised into focal assemblies tightly regulated and coordinated with other essential processes, such as DNA replication, and chromosomal segregation. Read more ....
Many of the functions and pathways involved in specific and recombinational repair are conserved in bacteria. Genetic, cytological and biochemical data allow us to postulate that during DSB repair, the RecN protein recognizes ssDNA tails on duplex DNA and specifically binds to the 3′-OH ends. Then, the AddAB complex, or RecJ together with RecQ or RecS, resect the 5′-ends, generating a proper substrate for RecA. The loading of RecA onto the 3′-ssDNA ends or onto SsbA-coated ssDNA relies on different avenues. We postulate that RecO interacts with RecR and/or RecL. These proteins alone or in concert with RecN might displace SsbA from ssDNA and promote RecA loading. Alternatively, after AddAB encounters the χ site, the complex is stably associated with the 3′-end of the χ site and may directly load RecA. RecU modulates RecA activities by promoting RecA-catalyzed strand invasion and by inhibiting RecA-mediated branch migration. RecA promotes partial disassembly of the RecN-induced large protein complex at the RC, and forms discrete threads or filaments that search for homology along the sister chromosome located in the other cell half. Then, RecF, and perhaps RecX, might contribute to RecA filament assembly/disassembly in an antagonistic manner, with RecX perhaps exerting a negative effect on the extension of RecA filaments. If a DSB occurs at the replication fork, RecA-medi-ated strand exchange creates a D-loop that PriA-DnaD-DnaB could exploit to assemble the DnaC–DnaI complex. Then, activated DnaC recruits DnaG and the chromosomal replicases. In any event, the branch migrating enzymes [RecG or RuvAB alone or in concert with RecV] promote strand exchange, and RecU catalyzes resolution of the HJ formed by recombination, and then the replication fork is fully re-established. Cytological studies have been used to demonstrate that at least the RecN, RecO, RecR, RecA, RecF or RecU proteins do not exist as a preassembled complex but rather assemble in an ordered fashion at the site of DSB. In particular the dynamic movements of RecA protein have been monitored in living cells.
The absence of RuvAB, RecV or RecG activities renders cells extremely sensitive to DNA damaging agents and results in a > 70-fold increase in the frequency of anucleate cells. These results suggest that these functions are required to avoid the accumulation of chromosomal dimers or dead-end recombination intermediates. In the absence of the RecA protein or in the absence of DNA homology, the NHEJ machinery might catalyze the joining of the two ends of a DSB, albeit at very low frequency, during vegetative growth, stationary phase or confer dry-heat resistance to dormant spores. Read more ....