Analysis of Break-Induced Replication

描述（由申请人提供）：断裂诱导复制（BIR），也称为重组依赖性DNA复制，在DNA复制和缺乏端粒酶的端粒维持中起着至关重要的作用。同源染色体间的BIR导致杂合性丢失（洛）。此外，BIR在异位同源序列之间的染色体重排的产生中是重要的，包括缺失、非相互易位和拷贝数变异。研究建议继续我们的详细的分子机制的BIR的分析，并表征修复复制叉的属性。第一个目的是使用HO核酸内切酶诱导的双链断裂（DSB）来启动异位BIR。实验研究了在BIR中具有不同于其在正常DNA复制中的作用的不同作用的关键蛋白，特别是Cdc 7激酶、非必需的Pod亚基、Pol 32和PCNA。一个新的PCNA等位基因，防止BIR，但不复制或基因转换的特点。Pol 32和PCNA中影响BIR的新突变将被寻求以定位受影响的结构域和蛋白质相互作用伙伴。Pol 32是否是DNA修复所必需的，也将被检查。第二个目的是关注在正常DNA复制的背景下特别出现的BIR。现在已经有可能在G1细胞中诱导位点特异性切口，这些切口将通过复制转化为DSB。将使用三种不同的方法：（1）经修饰的位点特异性核酸酶，在一条链上的Anil-K277 M，（2）在一条链上留下具有3 '-共价结合的Flp蛋白的切口的Flp-H305 L酶，和（3）切割32-bp位点以产生具有5'-连接的蛋白的切口的细菌RepC蛋白。这些系统使得第一次有可能在真实的时间过程中分析重组依赖的复制重新开始在一个破碎的复制叉。将通过遗传学和实时DNA分析检测姐妹染色单体修复和不均等姐妹染色单体交换。第三个目标转向产生拷贝数变异的机制的酵母模型，如现在称为MM-BIR的许多癌症中所见，但其细节尚未分析。DSB诱导的系统将检查染色体内和染色体间的模板开关，或者在高度分化的同源序列之间，或者在根本没有同源性的情况下，在这两种情况下，以创建一个可选择的基因，使我们能够获得甚至多个模板开关。 公共卫生相关性：断裂诱导复制（BIR）是一种DNA修复过程，在维持染色体复制过程中的完整性方面起着重要作用。BIR在维持其染色体末端而不重新激活端粒酶的癌症类别的永生化中是重要的。最近，BIR已经涉及产生基因拷贝数变异，也与肿瘤相关。这里提出的研究的目标是在一个定义良好的模型系统，允许详细分析的过程中研究BIR。

DESCRIPTION (provided by applicant): Break-induced replication (BIR), also called recombination-dependent DNA replication, plays critically important roles both during DNA replication and in the maintenance of telomeres lacking telomerase. BIR between homologous chromosomes leads to loss of heterozyosity (LOH). In addition, BIR is important in the creation of chromosome rearrangements between ectopic homologous sequences, including deletions, nonreciprocal translocations and copy number variation. Studies are proposed to continue our analysis of the detailed molecular mechanisms of BIR and to characterize the properties of a repair replication fork. The first Aim uses an HO endonuclease-induced double-strand break (DSB) to initiate ectopic BIR. Experiments examine key proteins in that have distinct roles in BIR different from their roles in normal DNA replication, especially the Cdc7 kinase, the nonessential Pold subunit, Pol32, and PCNA. A novel PCNA allele that prevents BIR but not replication or gene conversion will be characterized. New mutations in both Pol32 and PCNA that affect BIR will be sought to localize the domains and protein interaction partners that are affected. Whether Pol32 is required whenever Pold is indispensible for DNA repair will also be examined. A second Aim focuses attention on BIR that arises specifically in the context of normal DNA replication. It has now become possible to induce site-specific nicks in G1 cells that will be converted to DSBs by replication. Three different approaches will be used: (1) a modified site-specific nuclease, Ani1- K277M on one strand, (2)a Flp-H305L enzyme that leaves a nick with a 3'-covalently bound Flp protein on one strand, and (3) a bacterial RepC protein that cleaves a 32-bp site to produce a nick with a 5'-attached protein. These systems make it possible for the first time to analyze in real time process of recombination-dependent replication re-start at a broken replication fork. Sister-chromatid repair and unequal sister chromatid exchange will be tested, genetically and by real-time DNA analysis. The third Aim turns to a yeast model of the mechanism of generating copy number variation as seen in many cancers now called MM-BIR, but whose details have not been analyzed. A DSB-induced system will examine intra- and inter-chromosomal template switches either between highly diverged homeologous sequences or where there is no homology at all, in both cases to create a selectable gene that allows us to obtain even multiple template switches. PUBLIC HEALTH RELEVANCE: Break-Induced Replication (BIR) is a DNA repair process that plays important roles in the maintenance of the integrity of chromosomes during their replication. BIR is important in the immortalization of classes of cancer that maintain their chromosome ends without reactivating the telomerase enzyme. Most recently, BIR has been implicated in generating gene copy number variation, also associated with tumors. The goal of the research presented here is to study BIR in a well-defined model system that allows a detailed analysis of the process.