Investigation of DNA double strand break repair in Bacillus subtilis

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Although many proteins involved in repair of DNA double strand breaks (DSBs) have been identified (mostly in eukaryotic cells), it is still unclear how DSBs are repaired in detail. We have found that several proteins that are required for DNA recombination are important for DSB repair in the prokaryote Bacillus subtilis, among them the SMC like RecN protein. Additionally, we found that SbcC, the prokaryotic Rad50 ortholog, plays an important role in DSB repair, likewise to the SMC chromosome condensation complex. The function of these was highlighted by our finding that RecN, RecO and RecF are recruited to specific DSB repair centers (RCs) upon induction of even one specific DSB, with RecN organizing the RCs, and RecO recruiting RecF and most likely RecA. RecA formed inducible filaments extending from the RCs. RecNOF RCs formed independently of RecA, RecG or RecU suggesting that the latter proteins act downstream of RecNOF. Similarly, RecR and SbcC formed inducible foci, most likely being recruited into the observed RCs. RCs formed independently of the DNA polymerase machinery, and independently of ongoing replication. Thus, DSB repair can not only be visualized in live cells, but the pathway can be dissected using our system, which will shed light on the detailed function of many if not all proteins involved.