Break-induced replication and genome rearrangements

描述(由申请人提供)： DNA双链断裂(DSB)是一种细胞毒性损伤，在正常细胞代谢过程中自发发生，或通过DNA损伤剂处理细胞而发生。如果不修复或修复不当，DSB可能会导致突变事件，如染色体丢失、缺失、复制或易位，这些事件可能导致癌症发生。DSB的同源依赖修复通常通过一种保守的基因转换机制发生，防止广泛的杂合性丢失(LOH)或染色体重排。只有一端需要修复的断裂，例如在被侵蚀的无帽端粒上，或者当同源性被限制在DSB的一侧时，被认为是通过链侵入同源双链DNA，然后复制到染色体末端来修复的(断裂诱导复制，BIR)。由于来自DSB两端之一的BIR会导致广泛的LOH，这表明当DSB有两端时，BIR受到抑制，以便通过更保守的HR机制进行修复。此外，我们的研究表明，在BIR过程中形成的复制中间体是不稳定的，入侵端可以切换到不同的模板，从而导致易位。BIR和一种相关的机制，分叉停滞和模板切换(FoSTeS)，被认为是导致与人类疾病相关的非互惠易位和拷贝数变异的许多基因组重排的原因。这项建议的目标是了解BIR的机制，以及细胞如何从基因转换到BIR修复模式。具体目标是：(1)使用一种新的检测模板切换的基因测试来识别调控这一过程的基因。(2)确定对BIR期间出现的非互换易位使用异位模板是否存在偏见。(3)将用物理方法来确定与BIR相关的DNA合成是保守的还是非保守的，通过DNA梳理来确定分叉移动的速度，以及不同的DNA聚合酶和McM2-7复制解旋酶在BIR的启动和完成中的作用。 公共卫生相关性： 通过断裂诱导复制(BIR)修复DNA双链断裂可以导致与人类疾病相关的几种类型的染色体重排。BIR还参与在没有端粒酶的情况下维持端粒长度，这一过程在一些人类肿瘤中被激活。在这项建议中，将使用遗传和物理方法来确定在BIR过程中调节染色体重排的基因，并定义BIR中使用的DNA合成机制。

DESCRIPTION (provided by applicant): DNA double strand breaks (DSBs) are cytotoxic lesions that occur spontaneously during normal cell metabolism or by treatment of cells with DNA-damaging agents. If unrepaired or repaired inappropriately, DSBs can lead to mutagenic events, such as chromosome loss, deletions, duplications or translocations, events that can lead to carcinogenesis. The homology-dependent repair of DSBs usually occurs by a conservative gene conversion mechanism, preventing extensive loss of heterozygosity (LOH) or chromosome rearrangements. Breaks that present only one end for repair, for example at eroded uncapped telomeres or when homology is limited to one side of the DSB, are thought to repair by strand invasion into a homologous duplex DNA followed by replication to the chromosome end (break-induced replication, BIR). As BIR from one of the two ends of a DSB would result in extensive LOH it suggests BIR is suppressed when DSBs have two ends in order for repair to occur by a more conservative HR mechanism. Furthermore, our studies have shown that the replication intermediate formed during BIR is unstable and the invading end can switch to a different template resulting in a translocation. BIR and a related mechanism, fork stalling and template switching (FoSTeS), are thought to be responsible for many of the genome rearrangements that give rise to non-reciprocal translocations and copy number variation associated with human disease. The goals of this proposal are to understand the mechanisms of BIR and how cells switch from gene conversion to the BIR mode of repair. The specific aims are: (1) To use a new genetic assay that detects template switching to identify the genes that regulate this process. (2) To determine whether there is a bias in the use of ectopic templates for non-reciprocal translocations that arise during BIR. (3) Physical methods will be used to determine whether DNA synthesis associated with BIR is conservative or non-conservative, the rate of fork movement will be determined by DNA combing, and the role of different DNA polymerases and the Mcm2-7 replicative helicase in the initiation and completion of BIR will be determined. PUBLIC HEALTH RELEVANCE: The repair of DNA double-strand breaks by break-induced replication (BIR) can lead to several types of chromosome rearrangements that are associated with human disease. BIR is also involved in maintaining telomere length in the absence of telomerase and this process is activated in some human tumors. In this proposal, genetic and physical approaches will be used to identify the genes that regulate chromosome rearrangements during BIR, and to define the mechanism for DNA synthesis used in BIR.