Analysis of Break-Induced Replication

描述(申请人提供)：过去十年的研究表明，断裂诱导复制(BIR)的同源重组过程对于重新启动停滞或断裂的复制叉以及在没有端粒酶的情况下维持真核细胞端粒至关重要。此外，BIR在同源序列之间的染色体重排中似乎是重要的，包括缺失和非互换易位。在模式生物酿酒酵母中，有两个依赖于Rad52的、依赖于重组的DNA复制过程，一个需要RAD51重组酶，另一个利用Mre11-Rad50-Xrs2复合体和Rad59。这项建议侧重于更有效的、依赖RAD51的BIR途径。我们建议进行实验，以了解组装修复复制叉的详细分子机制和遗传要求，该复制叉可以复制至少几百kb的模板染色体。BIR产生非互换易位的单倍体菌株将被用于实时监测由巨核酸酶(如HO或L-SCEL)诱导的位点特异性双链断裂的修复。将确定BIR的DNA序列同源性要求，并将分析BIR进展的可能的序列特定障碍。DNA中的中间体和蛋白质到DSB修复部位的募集都可以实时跟踪。在建立和延长正常DNA复制中重要的蛋白质的作用将被评估它们在修复复制分叉的建立和进展中的作用，修复复制分叉可以在启动后的G1细胞或在没有竞争的正常复制的G2细胞中进行分析。将对修复复制叉进行分析，以了解其处理能力和保真度。同位素密度转移实验将被用来确定新复制的DNA链是从模板上移位还是保留为半保守的复制产物。最后，系统的基因组筛选将被用来识别在BIR和在端粒酶缺失的情况下维持端粒方面发挥关键作用的新基因。

DESCRIPTION (provided by applicant): Studies over the past decade have revealed that the homologous recombination process of Break-Induced Replication (BIR) is fundamentally important in re-starting stalled or broken replication forks as well as in maintaining eukaryotic telomeres in the absence of telomerase. In addition, BIR appears to be important in the creation of chromosome rearrangements between homologous sequences, including deletions and nonreciprocal translocations. In the model organism, Saccharomyces cerevisiae, there are two Rad52- dependent, recombination-dependent DNA replication processes, one of which requires the Rad51 recombinase and a second that utilizes the Mre11-Rad50-Xrs2 complex and Rad59. This proposal focuses on the more efficient, Rad51-dependent BIR pathway. Experiments are proposed to understand the detailed molecular mechanisms and genetic requirements to assemble a repair replication fork that can copy a template chromosome for at least several hundred kb. A haploid strain in which BIR produces a nonreciprocal translocation will be used to monitor in real time the repair of a site-specific double-strand break, induced by a meganuclease such as HO or l-Scel. The DNA sequence homology requirements for BIR will be determined and possible sequence-specific barriers to BIR progression will be analyzed. Both intermediates in the DNA and the recruitment of proteins to the site of DSB repair can be followed in real time. The role of proteins important in establishing and elongating normal DNA replication will be evaluated for their role in the establishment and progression of the repair replication fork, which can be analyzed in post-start G1 cells or in G2 cells, when there is no competing normal replication. The repair replication fork will be analyzed to learn about its processivity and fidelity. Isotope density transfer experiments will be used to determine if the newly replicated DNA strands are displaced from their template or remain as semi- conservative replication products. Finally, systematic genome screens will be used to identify new genes that play key roles in BIR and in telomere maintenance in the absence of telomerase.