Analysis of Break-Induced Replication

描述(由申请人提供)： 摘要染色体断裂的修复是维持染色体结构和基因组稳定性的关键。这一建议的重点是描述断裂诱导复制(BIR)修复双链染色体断裂(DSB)的机制。BIR在重新启动停滞和断裂的DNA复制叉、在没有端粒酶的情况下维持端粒以及新发现的单染色体断裂现象(称为染色质病)中发挥关键作用，这种现象与人类癌症和发育疾病有关。微同源介导的BIR(MM-BIR)过程被假设为解释导致远距离序列连接以创建新的基因融合的长距离模板切换事件。该实验室之前的工作已经确定了复制蛋白Pol32和增殖细胞核抗原的作用，它们对BIR是必不可少的，但对模式生物--发芽酵母中的正常复制或其他类型的DSB修复来说却不是。继续这项研究的拟议项目将侧重于两个主要专题。首先，将探索BIR的分子机制，通过可诱导的内切酶产生位点特异的DSB。已经开发了一种新的方法来研究BIR期间令人惊讶的频繁的模板跳跃，无论是在遥远位置的同源序列之间还是在同源序列之间(允许研究MM-BIR的高度失配的序列)。这种分析方法允许人们区分断裂的DNA末端通过链入侵定位并与远距离序列重组的方式，以及随后在BIR中复制的链如何跳到第三个位置。错配修复蛋白在阻止不同序列之间的BIR中的作用将被研究。将寻找模板跳跃使用与最初的链侵入步骤不同的机制的证据。跳跃不需要RAD51重组酶蛋白的可能性将被探索。将特别关注Rdh54的作用，它是模板跳跃所需的第一种蛋白质，但有能力 简单的BIR和其他重组事件。修复断裂的复制叉，通过用CRISPR内切酶划破特定的链，是否遵循与迄今研究的异位模型系统和端粒修复事件相同的规则，是一个根本重要的问题。第二个主要目标将是检查在修复连接处几乎没有同源性的移位和重排。将使用一种新的方法，该方法涉及通过连接远距离序列来创建功能内含子。最后，模板跳跃到不相关的序列将被恢复并通过DNA测序进行分析，以便更好地定义微同源性介导的事件需要多少同源性和相邻的同源性。这些研究对于理解与人类疾病相关的染色体重排的起源具有非常重要的意义，包括非互惠易位的形成所导致的杂合性丧失，节段性复制以及染色体中发生的染色体惊人的重排。

DESCRIPTION (provided by applicant): Summary The repair of broken chromosomes is essential for maintenance of chromosome structure and genome stability. This proposal focuses on delineating the mechanisms of repair of double-strand chromosome breaks (DSBs) by break-induced replication (BIR). BIR plays a key role in restarting stalled and broken DNA replication forks, in maintaining telomeres in the absence of the telomerase enzyme and in the newly discovered phenomenon of single chromosome shattering, known as chromothripsis, which is associated with human cancers and developmental diseases. A microhomology-mediated BIR (MM-BIR) process has been hypothesized to account for long-distance template switching events that lead to the joining of distant sequences to create novel gene fusions. Previous work from this laboratory has identified roles for replication proteins Pol32 and PCNA that are essential for BIR but not for normal replication or other types of DSB repair in the model organism, budding yeast. The proposed project to continue this study will focus on two main topics. First, the molecular mechanism of BIR will be pursued, creating site-specific DSBs by an inducible endonuclease. A novel assay has been developed to study surprisingly frequent template jumps during BIR both between homologous sequences in distant locations or between homeologous sequences (highly mismatched sequences that permit study of MM-BIR). This assay permits one to distinguish between the way in which a broken DNA end locates and recombines with a distant sequence by strand invasion and how the subsequent replicating strands in BIR then can jump to a third location. The role of mismatch repair proteins in discouraging BIR between diverged sequences will be investigated. Evidence that template jumping uses a different mechanism from the initial strand invasion step will be pursued. The possibility that the jump does not require the Rad51 recombinase protein will be explored. Special attention will be devoted to the role of Rdh54, the first protein that is specifically required for template jumps but competent for simple BIR and other recombination events. Whether repair of a broken replication fork, created by nicking a specific strand with a CRISPR endonuclease, obeys the same rules as the ectopic model systems and telomere-repair events that have so far been studied is a fundamentally important question. A second major goal will be to examine translocations and rearrangements in which there is little or no homology at the repair junctions. A novel assay involving creation o a functional intron by joining distant sequences will be used. Finally, template jumps into unrelated sequences will be recovered and analyzed by DNA sequencing in order to better define how much homology and adjacent homeology is required for microhomology-mediated events. These studies are highly significant in understanding the origins of chromosome rearrangements associated with human disease, including the loss of heterozygosity caused by the formation of nonreciprocal translocations, segmental duplications and the astonishing rearrangements in chromosomes experiencing chromothripsis.