Werner Syndrome Protein, DNA End Processing, and Double-Strand Break Repair

这项建议的目的是了解5‘链- 真核生物DNA双链断裂(DSB)的特异末端处理。DSB是 最有害的DNA损伤类型之一。如果没有正确维修，DSB 可能导致染色体缺失或易位，最终导致早产 细胞死亡或致癌转化。相应地，许多癌症易患疾病的基因， 例如Werner综合征蛋白(WRN)、Bloom综合征基因(BLM)、BRCA1和 BRCA2被认为与DSB修复有关。尽管进行了广泛的研究，但许多 关于DSB修复的基本机制问题仍然知之甚少。的 特别重要的是5‘链特异末端加工的机制 启动同源依赖的DSB修复。一种生化方法已经被用来 研究非洲爪哇卵提取液中DSB修复和DNA末端处理。单股 退火法(SSA)是一种同源依赖的DSB修复途径。 成功地重组并被证明依赖于非洲爪哇 综合征蛋白(XWRN)。进一步的分析揭示了一种新的END机制 正在处理。末端首先被RecQ型DNA解旋酶解开，主要是xWRN， 然后，5‘ss-Tail被5’-gt；3‘ss-DNA外切酶降解，主要是非洲爪哇 与DNA2(XDNA2)同源，最终产物为3‘SS-Tail。建立在这些基础上 进展，提出了两个具体目标，以更全面地调查 从三种酶的活性表征DNA末端加工的机制 关键末端加工蛋白及其缺失对末端加工的影响 非洲爪哇卵萃取物。在特定目的I中，EXO1(XEXO1)的非洲爪哇同系物将 对其进行研究以确定其在末端加工中的机械作用。核酸酶活性的测定 XEXO1的特性及其对非洲爪哇卵提取液最终加工的影响 将被分析以确定xEXO1是作用于ss-DNA(类似于xDNA2)还是作用于ds-DNA DNA(与xDNA2不同)和IF xWRN调控xEXO1途径。具体而言 目的研究Mre11(XMRE11)和CtIP1(XCtIP1)的非洲爪哇同源物 以确定它们在末端加工中的机械作用。具体地说， XMRE11的S核酸酶活性，引发了许多矛盾和困惑 在其他系统中的观测，将被严格地剖析。总而言之，这些研究将 帮助我们阐明DSB最基本但最不了解的流程之一 两种临床上重要的蛋白质在基因组维持中的修复和功能。

The objective of this proposal is to understand the mechanism for the 5' strand- specific end processing of DNA double-strand breaks (DSBs) in eukaryotes. DSBs are among the most deleterious types of DNA damages. If not properly repaired, DSBs might cause chromosome deletions or translocations, ultimately leading to premature cell death or oncogenic transformation. Accordingly, many cancer-prone disease genes, such as Werner syndrome protein (WRN), Bloom syndrome gene (BLM), BRCA1, and BRCA2, have been implicated in DSB repair. Despite extensive research, many fundamental mechanistic questions about DSB repair are still poorly understood. Of particular importance is the mechanism for the 5' strand-specific end processing that initiates homology-dependent DSB repair. A biochemical approach has been taken to study DSB repair and DNA end processing in Xenopus egg extracts. Single-strand annealing (SSA), one of the homology-dependent DSB repair pathways, has been successfully reconstituted and shown to be dependent on the Xenopus Werner syndrome protein (xWRN). Further analysis has revealed a novel mechanism for end processing. The end is first unwound by a RecQ-type DNA helicase, mainly xWRN, the 5' ss-tail is then degraded by a 5' -> 3' ss-DNA exonuclease, mainly the Xenopus homologue of DNA2 (xDNA2), and the final product is a 3' ss-tail. Building on these advances, two specific aims are proposed to more comprehensively investigate the mechanism of DNA end processing by characterizing the enzymatic activities of three key end processing proteins and analyzing how their depletions affect end processing in Xenopus egg extracts. In specific aim I, the Xenopus homologue of EXO1 (xEXO1) will be studied to determine its mechanistic role in end processing. The nuclease activity of xEXO1 will be characterized and its effect on end processing in Xenopus egg extracts will be analyzed to determine if xEXO1 acts on ss-DNA (similarly to xDNA2) or on ds- DNA (distinctively from xDNA2) and if xWRN modulates the xEXO1 pathway. In specific aim II, the Xenopus homologues of MRE11 (xMRE11) and CtIP1 (xCtIP1) will be studied to determine their mechanistic roles in end processing. In particular, the role of xMRE11's nuclease activity, which has given rise to many conflicting and confusing observations in other systems, will be rigorously dissected. Together, these studies will help us elucidate one of the most fundamental but least understood processes for DSB repair and the function of two clinically important proteins in genome maintenance.