Study of Break-induced Replication in Mammalian Cells

大体染色体重排(GCR)是癌症和DNA双重感染的标志 链断裂(DSB)是GCR的主要来源。许多GCR活动都涉及 复制过程，但潜在的机制尚不清楚。基于对酵母的研究， 一种称为断裂诱导复制(BIR)的DSB修复途径被认为是一种主要的 复制GCR的来源。由于对生物红外光谱的机理研究主要集中在机理上 在酵母中，研究BIR机制及其在抑制基因组中的作用的模型系统 哺乳动物细胞的不稳定性是非常需要的。 我们已经建立了一种基于绿色荧光蛋白的新型BIR报告程序，使其能够 哺乳动物BIR机制的系统研究及其遗传成分分析 细胞。我们假设，BIR一方面参与了DSB修复，以防止 染色体断裂并有助于维持基因组的稳定性，但另一方面， 是高度诱变的，它的失调将导致基因组的不稳定。在这 建议，我们将研究复制和致癌应激如何诱导BIR和 导致基因组不稳定。我们还将研究其作用和潜在机制 BIR中的复制解旋酶，如Pif1和其他复制蛋白。我们会破译 ATR介导的磷酸化在促进BIR和检测功能中的作用 复制蛋白与RAD51在折叠叉处的相互作用以启动复制的BIR 重新启动。我们的研究将阐明BIR是如何在哺乳动物细胞中运作的，并提供 关于维持基因组完整性的新的机械论见解。它还将打开新的 探索癌症治疗的BIR途径的机会。

Gross chromosomal rearrangement (GCR) is a hallmark of cancer and DNA double- strand breaks (DSBs) constitute a major source of GCR. Many GCR events involve a replicative process, but the underlying mechanism is not clear. Based on the study in yeast, a DSB repair pathway called break-induced replication (BIR) is believed to be a major source of replicative GCR. Since the mechanistic study of BIR has been mostly carried out in yeast, a model system to study BIR mechanism and its role in suppressing genome instability in mammalian cells is highly in demand. We have established a novel EGFP-based BIR reporter, making it possible to systematically study BIR mechanisms and analyze its genetic components in mammalian cells. We hypothesize that BIR, on one hand, is involved in DSB repair to prevent chromosome breakages and help maintenance of genome stability, but on the other hand, is highly mutagenic and its dysregulation would contribute to genome instability. In this proposal, we will study how BIR is induced by replication and oncogenic stress and contributes to genome instability. We will also study the role and the underlying mechanism of replication helicases such as Pif1 and other replication proteins in BIR. We will decipher the role of ATR-mediated phosphorylation in promoting BIR and examine functional interplay of replication proteins with Rad51 at collapsed forks to initiate BIR for replication restart. Our study will shed light on how BIR is operated in mammalian cells and provide new mechanistic insight on the maintenance of genome integrity. It will also open new opportunities to explore the BIR pathway for cancer treatment.