Replication fork repair

描述(申请人提供)：重组修复对于细胞存活和遗传物质的稳定性很重要。在人类中，修复效率低下与癌症倾向、神经和发育缺陷以及过早衰老有关。最近的证据表明，在所有细胞中，每一轮复制都需要某种形式的复制叉修复。在这项拟议的研究中，我们将解决原核和真核重组中的几个重要问题，这些问题与每个生物体的复制叉修复有关。(1)RecA Paralog蛋白如何促进重组？每种生物似乎都使用一个RecA-/RAD51同源链交换蛋白和至少一个其他RecA/RAD51平行蛋白。Paralog们在做什么？我们将使用生物化学和遗传学相结合的方法来解决RecA Paralog蛋白，RADA/SMS在大肠杆菌遗传重组中的作用。(2)哪些分支DNA分子是重组的中间产物，哪些酶可以分解它们？我们对处理重组的分支DNA中间体的酶的了解是不完整的。我们将检测大肠杆菌RuvC相关蛋白YqgF是否参与重组修复，并能够催化重组机制预测的分支分子的裂解。(3)模板开关修复的中介因素是什么？这是一种重组机制，导致姐妹染色体交换，而不需要链交换蛋白，如RecA。我们之前的工作确定了参与大肠杆菌模板切换修复的前两个因素，即DNA聚合酶III的伴侣DNAK和伽马/tau亚单位DNAX。还有什么其他因素促成了这种反应？利用遗传分析，我们将测试复制体蛋白质的参与，并寻找目前未知的因素。复制叉修复对于细胞的存活和基因的稳定是重要的。在人类中，修复效率低下与癌症倾向、神经和发育缺陷以及过早衰老有关。这项提议试图了解复制叉子修复的机制。

DESCRIPTION (provided by applicant): Recombinational repair is important for cell survival and stability of genetic material. In humans, inefficiency of repair has been associated with cancer proneness, neurological and developmental defects and premature aging. Recent evidence suggests that, in all cells, every round of replication requires some form of replication fork repair. In this proposed study, we will address several important questions in prokaryotic and eukaryotic recombination that have relevance to replication fork repair in every organism. (1) How do the RecA paralog proteins facilitate recombination? Every organism appears to employ a RecA-/Rad51 orthologous strand exchange protein and at least one other RecA/Rad51 paralog protein. What are the paralogs doing? We will use a combined biochemical and genetic approach to address the role of the RecA paralog protein, RadA/Sms in genetic recombination of E. coli. (2) What branched DNA molecules are intermediates of recombination and which enzymes resolve them? Our knowledge of the enzymes that process branched DNA intermediates of recombination is incomplete. We will assay whether the RuvC-related protein of E. coli, YqgF, is involved in recombinational repair and can catalyze cleavage of branched molecules predicted by recombination mechanisms. (3) What factors mediate template-switch repair? This is a recombinational mechanism that leads to sister chromosome exchange without the requirement for a strand-exchange protein such as RecA. Our previous work identified the first two factors involved in template-switch repair of E. coli, the chaperone DnaK and the gamma/tau subunit of DNA polymerase III, DnaX. What other factors enable this reaction? Using genetic analysis, we will test the involvement of proteins of the replisome and seek factors presently unknown. Replication fork repair is important for cell survival and stability of genes. In humans, inefficiency of repair has been associated with cancer proneness, neurological and developmental defects and premature aging. This proposal seeks to understand the mechanisms of replication fork repair.