Regulation of stalled fork repair in mammalian cells

哺乳动物细胞中停滞叉修复的调节

• 批准号: 10434669
• 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
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-03 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10434669
• 关键词: Acute Myelocytic Leukemia; Affect; Anemia; BRCA1 gene; BRCA2 gene; Binding; Biological Assay; Bone marrow failure; 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' 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; 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; brca gene; 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

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.

项目总结 在复制分叉停滞处启动的无错误DNA修复是预防基因组的关键 细胞循环中的不稳定性。停滞的叉子修复缺陷直接与癌症和其他人类疾病有关 疾病。Fanconi贫血(FA)是一种罕见的常染色体隐性遗传(或X连锁)疾病，由失活的 几种FA基因中的任何一种。FA的临床表现包括儿童贫血和进行性贫血。 骨髓衰竭，加上身材矮小和先天性缺陷，影响到各种器官。这个 癌症的风险，包括实体瘤，特别是急性骨髓性疾病的发病率很高。 白血病。编码核酸酶协调支架蛋白SLX4/FANCP的基因在 一些患有Fanconi贫血的人，并因与 核酸酶MUS81、XPF和SLX1。我们采用了大肠杆菌Tus/Ter复制叉阻滞 在哺乳动物细胞中使用的复合体，并用它来刺激特定部位的复制叉子停滞和 哺乳动物染色体上特定位置的同源重组(HR)。我们发现SLX4扮演着一个 在介导TUS/Ter诱导的无错误心率中起关键作用。此功能仅限于停顿的叉子维修和 不是由传统的染色体双链断裂引起的HR的特征。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如何调控同源重组 复制分叉停滞。我们将使用物理和基因组分析来测量特定的DNA结构， 在TUS/Ter停滞的叉子上形成，并将决定SLX4是否调节 这些DNA结构。该项目将确定SLX4协调失速叉子的机制 在面临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。