Investigation of DNA double strand break repair in Bacillus subtilis

虽然已经鉴定了许多参与DNA双链断裂（DSB）修复的蛋白质（大多数在真核细胞中），但DSB是如何详细修复的仍然不清楚。我们已经发现，在原核生物枯草芽孢杆菌中，DNA重组所需的几种蛋白质对于DSB修复是重要的，其中包括SMC样RecN蛋白。此外，我们发现，SbcC，原核Rad50直系同源物，在DSB修复中起着重要的作用，同样的SMC染色体凝聚复合物。我们的发现强调了这些的功能，即RecN，RecO和RecF在诱导甚至一个特定的DSB时被招募到特定的DSB修复中心（RC），RecN组织RC，RecO招募RecF和最有可能的RecA。RecA形成从RC延伸的诱导丝。RecNOF RC独立于RecA、RecG或RecU形成，表明后者蛋白在RecNOF下游起作用。类似地，RecR和SbcC形成诱导灶，最有可能被募集到观察到的RC中。RC的形成独立于DNA聚合酶机制，并且独立于正在进行的复制。因此，DSB修复不仅可以在活细胞中可视化，而且可以使用我们的系统解剖该途径，这将揭示许多（如果不是所有）相关蛋白质的详细功能。

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.