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

许多参与DNA双链断裂(DSB)修复的蛋白质已被鉴定，并对它们的生化性质进行了不同程度的研究。然而，DSB修复的确切途径仍不清楚。我们最近已经能够证明在枯草芽孢杆菌细胞中存在对DSB反应而瞬时诱导的修复中心(RCS)，并能够可视化几种蛋白质在RCS中的临时和有序的募集。这些实验已经为DSB修复提供了重要的新见解，并且是剖析活细胞中DSB修复蛋白功能的一个很好的系统。这项建议涉及修复蛋白RecJ、RecX、YhaN/O、SBCC/D、radC和SMF，它们发挥着重要的功能，但方式截然不同。RecJ外切酶在DSB修复的早期时间点起作用，但像RADC一样，也在非应激细胞的复制叉处起作用。RecX已被证明与主要修复蛋白RecA相互作用，并可能调节RecA的活性。YhaN和SBCC是SMC样蛋白，其功能尚不清楚。我们计划从基因上剖析这些基因的功能，通过将这些缺失与所有其他已知的DSB基因缺失相结合，通过蛋白质纯化和生化分析，以及通过可视化GFP融合，将蛋白质放入修复过程中正确的外观和组装环境中。

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.