Regulation of stalled fork repair in mammalian cells

哺乳动物细胞中停滞叉修复的调节

基本信息

批准号：
10006891
负责人：
Ralph Scully
金额：
$ 35万
依托单位：
BETH ISRAEL DEACONESS MEDICAL CENTER
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：
2019-09-03 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10006891
关键词：
Acute Myelocytic Leukemia Affect Anemia BRCA1 gene BRCA2 gene Binding Biological Assay CRISPR/Cas technology Cell Cycle Cells Childhood Clinical Complement Complex Congenital Abnormality Coupled DNA DNA Damage DNA Repair DNA Replication Damage DNA Structure DNA biosynthesis DNA metabolism DNA replication fork Defect Disease Escherichia coli Fanconi&apos s Anemia Gene Conversion Genes Genetic Genome Stability Genomic Instability Genomics Hereditary Breast and Ovarian Cancer Syndrome Incidence Individual Knock-out Link Malignant Neoplasms Malignant neoplasm of ovary Mammalian Cell Mammalian Chromosomes Measures Mediating Molecular Mus Mutate Mutation Nonhomologous DNA End Joining Organ Outcome Pancytopenia Pathologic Pathway interactions Pattern Phenotype Physiological Play Prevention Process Regulation Replication-Associated Process Reporter Role Scaffolding Protein Site Solid Neoplasm Structure Surgical incisions Susceptibility Gene Time Ursidae Family Work Yeasts cancer genome cancer risk cancer therapy chromatin immunoprecipitation defined contribution developmental disease embryonic stem cell endodeoxyribonuclease SceI endonuclease homologous recombination human disease insight malignant breast neoplasm mutant new therapeutic target novel nuclease physiologic model preservation prevent repaired replication stress scaffold success targeted cancer therapy tool

项目摘要

PROJECT SUMMARY Error-free DNA repair initiated at the sites of replication fork stalling is critical to the prevention of genomic instability in cycling cells. Defects in stalled fork repair have been directly implicated in cancer and other human diseases. Fanconi Anemia (FA) is a rare, autosomal recessive (or X-linked) disease caused by inactivation of any one of several FA genes. The clinical manifestations of FA include childhood anemia and progressive bone marrow failure, together with short stature and congenital defects affecting a wide variety of organs. The risk of cancer, including solid tumors, is elevated, with particularly high incidence of acute myelogenous leukemia. The gene encoding a nuclease-coordinating scaffolding protein, SLX4/FANCP, is found mutated in some individuals with Fanconi anemia and has been implicated in stalled fork repair through interactions with the nucleases MUS81, XPF and SLX1. We adapted the Escherichia coli Tus/Ter replication fork arrest complex for use in mammalian cells and have used it to provoke site-specific replication fork stalling and homologous recombination (HR) at defined loci of a mammalian chromosome. We find that SLX4 plays a crucial role in mediating error-free HR induced by Tus/Ter. This function is restricted to stalled fork repair and is not a feature of HR induced by a conventional chromosomal double strand break. In work proposed here, we will use novel tools developed by the Scully lab, to analyze how SLX4 regulates homologous recombination at stalled replication forks. We will use physical and genomic assays to measure specific DNA structures that form at the Tus/Ter-stalled fork and will determine whether SLX4 regulates the formation or metabolism of these DNA structures. This project will identify the mechanisms by which SLX4 coordinates stalled fork processing to preserve genome stability in the face of replication stress. Success in this work will lead to the identification of new targets for therapy in cancer and other human diseases.

项目概要在复制叉停滞位点启动的无错误 DNA 修复对于预防基因组复制至关重要循环细胞的不稳定性。停滞叉修复缺陷与癌症和其他人类疾病直接相关疾病。范可尼贫血 (FA) 是一种罕见的常染色体隐性遗传（或 X 连锁）疾病，由多个 FA 基因中的任何一个。 FA 的临床表现包括儿童期贫血和进行性贫血。骨髓衰竭，以及影响多种器官的身材矮小和先天性缺陷。这癌症（包括实体瘤）的风险升高，其中急性骨髓性肿瘤的发病率特别高白血病。编码核酸酶协调支架蛋白 SLX4/FANCP 的基因在一些患有范可尼贫血的人，通过与以下因素的相互作用而参与了停滞的叉修复：核酸酶 MUS81、XPF 和 SLX1。我们改造了大肠杆菌 Tus/Ter 复制叉停滞用于哺乳动物细胞的复合物，并用它来引发位点特异性复制叉停滞和哺乳动物染色体指定位点上的同源重组 (HR)。我们发现SLX4起到了在介导 Tus/Ter 诱导的无差错 HR 中发挥着至关重要的作用。此功能仅限于修复失速的货叉不是传统染色体双链断裂诱导的 HR 特征。在这里提出的工作中，我们将使用Scully实验室开发的新工具来分析SLX4如何调节同源重组复制叉停滞。我们将使用物理和基因组检测来测量特定的 DNA 结构，在 Tus/Ter 失速叉处形成，并将决定 SLX4 是否调节这些DNA结构。该项目将确定 SLX4 协调失速前叉的机制面对复制压力时保持基因组稳定性的处理。这项工作的成功将导致确定癌症和其他人类疾病治疗的新靶点。