Choreography of the assembly of proteins in DNA double strand break repair centres in bacteria

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Many proteins involved in the repair of double strand breaks (DSBs) in DNA have been identified, and their biochemical properties have been studied to various degrees. However, the exact pathway of DSB repair is still unclear. We have recently been able to prove the existence of repair centres (RCs) that are transiently induced in response to DSBs in Bacillus subtilis cells, and to visualize the temporal and ordered recruitment of several proteins to RCs. These experiments have already provided important novel insight into DSB repair, and are an excellent system to dissect the function of DSB repair proteins in live cells. This proposal deals with repair proteins RecJ, RecX, YhaN/O, SbcC/D, RadC and Smf, which perform important functions, but in very different ways. The RecJ exonuclease functions at an early time point during DSB repair, but, like RadC, also at the replication fork in non-stressed cells. RecX has been shown to interact with the major repair protein RecA, and may modulate RecA activity. YhaN and SbcC are SMC-like proteins whose function is yet unclear. We plan to dissect the function of the genes genetically, by combining the deletions with all other known deletions in DSB genes, by protein purification and biochemical analysis, and by visualization of GFP fusions, to put the proteins into the correct context of appearance and assembly during repair.