Analysis of Break-Induced Replication

描述(申请人提供)：断裂诱导复制(BIR)，也被称为重组依赖的DNA复制，在DNA复制和缺乏端粒酶的端粒的维持中发挥着至关重要的作用。同源染色体间的BIR导致杂合性丢失(LOH)。此外，BIR在异位同源序列之间的染色体重排中也是重要的，包括缺失、非互换易位和拷贝数变异。建议进行研究，继续分析BIR的详细分子机制，并表征修复复制叉子的特性。第一个目的是使用HO内切酶诱导的双链断裂(DSB)来启动异位BIR。实验检测了在BIR中具有不同于其在正常DNA复制中的角色的关键蛋白，特别是CDC7激酶、非必需的Pold亚基、Pol32和PCNA。一个新的增殖细胞核抗原等位基因，可以防止BIR，但不能复制或基因转换将被描述。将寻求Pol32和增殖细胞核抗原中影响BIR的新突变来定位受影响的结构域和蛋白质相互作用伙伴。当Pold对于DNA修复是必不可少的时，是否需要Pol32也将被检查。第二个目的是将注意力集中在BIR上，这是在正常DNA复制的背景下特别出现的。现在已经有可能在G1细胞中诱导特定部位的缺口，并通过复制将其转化为DSB。将使用三种不同的方法：(1)修饰的位点特异性核酸酶，Ani1-K277M在一条链上，(2)FLP-H305L酶，在一条链上留下一个3‘-共价结合的FLP蛋白，以及(3)一个细菌RepC蛋白，它切割一个32bp的位点，产生一个带有5’-连接蛋白的缺口。这些系统首次使实时分析依赖于重组的复制在断裂的复制叉处重新开始的过程成为可能。姐妹染色单体修复和不平等姐妹染色单体交换将通过遗传和实时DNA分析进行测试。第三个目标转向一个酵母模型，该模型描述了在许多癌症中发现的拷贝数变异的产生机制，现在被称为MM-BIR，但其细节尚未被分析。DSB诱导的系统将检查染色体内和染色体间的模板开关，要么是在高度分化的同源序列之间，要么是在根本没有同源性的地方，在这两种情况下，都会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，在复制过程中维持染色体的完整性起着重要作用。BIR在不重新激活端粒酶的情况下保持染色体末端的癌症类别的不朽中是重要的。最近，BIR被认为与基因拷贝数变异有关，这也与肿瘤有关。这里提出的研究目标是在一个定义明确的模型系统中研究BIR，以便能够对过程进行详细分析。

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.