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

本提案的目的是了解5'链的机制- 真核生物中DNA双链断裂（DSB）的特异性末端加工。DSB是 是最有害的DNA损伤类型之一如果没有得到适当的修复， 可能会导致染色体缺失或易位，最终导致过早 细胞死亡或致癌转化。因此，许多癌症易感基因， 如沃纳综合征蛋白（WRN）、布卢姆综合征基因（BLM）、BRCA 1和 BRCA 2与DSB修复有关。尽管进行了广泛的研究，许多 关于DSB修复的基本机制问题仍然知之甚少。的 特别重要的是用于5 ′链特异性末端加工的机制， 启动同源依赖性DSB修复。已经采取了一种生物化学方法， 研究爪蟾卵提取物中DSB修复和DNA末端加工。单链 退火（SSA），同源依赖性DSB修复途径之一，已被 成功地重建并显示出依赖于爪蟾沃纳 综合征蛋白（xWRN）。进一步的分析揭示了一种新的机制， 处理.末端首先被RecQ型DNA解旋酶解旋，主要是xWRN， 5' ss-tail然后被5' -> 3' ss-DNA外切核酸酶降解，主要是非洲爪蟾 DNA 2的同源物（xDNA 2），并且最终产物是3'ss-尾。根据这些 进步，提出了两个具体目标，以更全面地调查 DNA末端加工的机制，通过表征三种酶的酶活性， 关键末端加工蛋白质，并分析它们的消耗如何影响末端加工， 爪蟾卵提取物。在具体目标I中，EXO 1的爪蟾同源物（xEXO 1）将 进行研究，以确定其在最终加工中的机械作用。的核酸酶活性 xEXO 1将被表征，并将其对非洲爪蟾卵提取物最终加工的影响 将被分析以确定xEXO 1是否作用于ss-DNA（类似于xDNA 2）或ds-DNA。 DNA（与xDNA 2不同）以及xWRN是否调节xEXO 1途径。在特定 目的二是研究MRE 11（xMRE 11）和CtIP 1（xCtIP 1）的爪蟾同源物 以确定它们在最终加工中的机械作用。特别是， xMRE 11的核酸酶活性，这引起了许多矛盾和混乱， 在其他系统中的观察，将被严格剖析。这些研究将 帮助我们阐明DSB最基本但最不了解的过程之一 修复和两个临床上重要的蛋白质在基因组维护中的功能。

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.