Study of Break-induced Replication in Mammalian Cells

哺乳动物细胞断裂诱导复制的研究

基本信息

批准号：
10528444
负责人：
Xiaohua Wu
金额：
$ 49.39万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-12-10 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10528444
关键词：
Abbreviations Affect Aphidicolin Applications Grants Biological Models CCNE1 gene Characteristics Chromosomal Rearrangement Chromosome Breakage Chromosomes Complex Copy Number Polymorphism DNA Damage DNA Double Strand Break DNA Repair DNA biosynthesis DNA replication fork Defect Dependence Double Strand Break Repair Event Exhibits Functional disorder Gene Conversion Genetic Genetic Diseases Genome Stability Genomic Instability Immunoprecipitation Link Loss of Heterozygosity MCM10 gene MCM2 gene Maintenance Malignant Neoplasms Mammalian Cell Mediating Mitotic Modification Mutate Mutation Nonhomologous DNA End Joining Oncogenes Oncogenic Pathologic Mutagenesis Pathway interactions Phosphorylation Play Process Proteins RAS genes Rad51 recombinase Reporter Role Single Nucleotide Polymorphism Source Stress Yeasts cancer cell cancer therapy genome integrity helicase homologous recombination hydroxyurea insight malignant breast neoplasm mutant neoplastic cell novel prevent recruit repaired replication stress response tandem mass spectrometry telomere tumorigenesis

项目摘要

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.

染色体总重排 (GCR) 是癌症和 DNA 双链的一个标志。链断裂 (DSB) 是 GCR 的主要来源。许多 GCR 活动都涉及复制过程，但其潜在机制尚不清楚。基于对酵母的研究，称为断裂诱导复制 (BIR) 的 DSB 修复途径被认为是主要的修复途径。复制性 GCR 的来源。由于 BIR 的机理研究已大部分开展在酵母中，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。我们建立了一种新型的基于 EGFP 的 BIR 报告器，使得系统研究哺乳动物BIR机制并分析其遗传成分细胞。我们假设 BIR 一方面参与 DSB 修复以防止染色体断裂有助于维持基因组稳定性，但另一方面，具有高度致突变性，其失调会导致基因组不稳定。在这个提案中，我们将研究复制和致癌应激如何诱导 BIR 以及导致基因组不稳定。我们还将研究其作用和底层机制复制解旋酶，例如 Pif1 和 BIR 中的其他复制蛋白。我们将破译 ATR 介导的磷酸化在促进 BIR 中的作用并检查功能复制蛋白与塌陷叉处的 Rad51 相互作用，启动 BIR 进行复制重新启动。我们的研究将揭示 BIR 在哺乳动物细胞中的运作方式，并提供关于维持基因组完整性的新机制见解。也将开启新的探索癌症治疗的 BIR 途径的机会。