Study of Break-induced Replication in Mammalian Cells

总染色体重排（GCR）是癌症和DNA双链的标志。 链断裂（DSB）构成GCR的主要来源。许多GCR事件涉及 复制过程，但其内在机制尚不清楚。基于对酵母的研究， 被称为断裂诱导复制（BIR）的DSB修复途径被认为是 复制性GCR的来源。由于对BIR机理的研究已基本完成， 在酵母中，研究BIR机制及其在抑制基因组中作用的模型系统 哺乳动物细胞中的不稳定性是高度需要的。 我们已经建立了一种新的基于EGFP的BIR报告基因， 系统地研究了哺乳动物BIR机制并分析了其遗传组成 细胞我们假设，一方面，BIR参与DSB修复， 染色体断裂，有助于维持基因组的稳定性，但另一方面， 是高度致突变的，其失调将导致基因组不稳定。在这 建议，我们将研究如何BIR是由复制和致癌应激诱导， 导致基因组不稳定。我们亦会研究这两个因素的作用及背后的机制 复制解旋酶，如Pif1和其他复制蛋白在BIR。我们将破译 ATR介导磷酸化在促进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BIR用于复制 重启我们的研究将阐明BIR如何在哺乳动物细胞中运作， 关于维持基因组完整性的新的机械见解。它还将开启新的 探索BIR途径治疗癌症的机会。

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.