Functions of human RAD51 and its paralogs during DNA interstrand crosslink repair

人类 RAD51 及其旁系同源物在 DNA 链间交联修复过程中的功能

• 批准号: 9399639
• 负责人: Agata Smogorzewska
• 金额: $ 42.54万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9399639
• 关键词: Acute; Affect; Anemia; BRCA2 Mutation; BRCA2 Protein; BRCA2 gene; Behavior; Binding; Biological Models; Bone Marrow; Breast; Cause of Death; Cell Line; Cell physiology; Cells; Chromosome Breakage; Chromosome abnormality; Complement; Complex; DNA; DNA Crosslinking; DNA Damage; DNA Interstrand Crosslinking; DNA Markers; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA damage checkpoint; DNA replication fork; Data; Defect; Development; Double Strand Break Repair; Electron Microscopy; Environment; Fanconi Anemia pathway; Fanconi' s Anemia; Fibroblasts; Functional disorder; Genes; Genetic; Genetic Diseases; Genetic Recombination; Genetic Transcription; Genome Stability; Genotoxic Stress; Goals; Head and Neck Cancer; Human; Hypersensitivity; International; Ionizing radiation; Kinetics; Knock-out; Knowledge; Lead; Light; Link; Malignant Female Reproductive System Neoplasm; Malignant Neoplasms; Malignant neoplasm of ovary; Mediating; Mitomycins; Mus; Mutation; Myelogenous; Ovary; Pancreas; Pancytopenia; Patients; Pharmaceutical Preparations; Predisposition; Prevention; Process; Proteins; RAD51C gene; Rad51 recombinase; Rare Diseases; Reaction; Recombinants; Registries; Research; Resolution; Role; Sister Chromatid Exchange; Site; Stem cells; Structure; System; Testing; Thumb structure; Toxin; Translating; Tumor Suppression; Tumor Suppressor Proteins; United States; XRCC2 gene; Xenopus; base; cancer therapy; chemotherapy; crosslink; ds-DNA; egg; embryonic stem cell; exome sequencing; experimental study; homologous recombination; in vitro Assay; malignant breast neoplasm; mutant; novel strategies; nuclease; paralogous gene; prevent; recessive genetic trait; recombinase; recombinational repair; recruit; repaired; response; tool; tumorigenesis

Cancer development is greatly accelerated when DNA is not properly repaired during replication. Naturally occurring mutations in patients with Fanconi anemia have delineated a DNA repair pathway that is responsible for assuring genomic stability and preventing cancer formation. Fanconi anemia, a recessive genetic disease is characterized by a very high predisposition to developing cancer including acute myelogenous anemia, head and neck cancers, and gynecologic cancers. In addition, monoallelic mutations in the Fanconi anemia genes BRCA2 (FANCD1), PALB2 (FANCN), BRIP1 (FANCJ), and RAD51C (FANCO), predispose to breast and ovarian cancer. Cells derived from Fanconi anemia patients lack the ability to repair a very toxic type of DNA damage, interstrand crosslink, which covalently links the Watson and Crick strands of DNA together precluding their separation. This type of DNA damage may be caused by toxins in the environment, cellular processes metabolites or by chemotherapy during cancer treatment. Using a whole exome sequencing of a patient in the International Fanconi Anemia Registry, we have identified a mutation in a key cellular recombinase, RAD51. The patient was born with developmental abnormalities including an absent thumb. Bone marrow function is normal to date. Patient fibroblasts are hypersensitive to crosslinking agents but the chromosomal breakage levels following crosslinking agent exposure are only mildly elevated compared to the much higher levels seen in other Fanconi anemia patient fibroblasts. Although RAD51 foci formation is reduced and delayed following treatment with a range of DNA damaging agents, the patient fibroblasts do not exhibit sensitivity to ionizing radiation. In addition, the patient fibroblasts show wild type levels of sister chromatid exchanges following treatment with a DNA crosslinking drug and are capable of performing homologous recombination for repairing dysfunctional GFP gene. Collectively, these results suggest homology dependent repair of double strand breaks is not impaired in the patient cells. Additional experiments suggest that the essential function of RAD51 at the sites of stalled replication forks and quite possibly the key function necessary for prevention of tumorigenesis is to protect the DNA from being degraded, precluding correct repair. In the proposed experiments, we strive to understand the mechanism of function of RAD51 and its binding partners during crosslink repair. First, we propose to use the patient cell lines to understand homologous recombination-independent function of RAD51 and BRCA2 during DNA interstrand crosslink repair. Secondly, we want to understand the interplay between RAD51 and the Fanconi anemia pathway. Finally, we will study how the RAD51-interacting proteins affect the RAD51 function at the stalled replication fork. The overarching goal is to understand how the RAD51 and the associated factors might be preventing tumorigenesis by functioning during unperturbed DNA replication.

当DNA在复制过程中没有正确修复时，癌症的发展会大大加速。范可尼贫血患者中自然发生的突变描述了一种DNA修复途径，该途径负责确保基因组稳定性和预防癌症形成。范可尼贫血是一种隐性遗传病，其特征是非常高的发生癌症的倾向，包括急性骨髓性贫血、头颈癌和妇科癌症。此外，范可尼贫血基因BRCA2（FANCD 1）、PALB 2（FANCN）、BRIP 1（FANCJ）和RAD 51 C（FANCO）的单等位基因突变易患乳腺癌和卵巢癌。来自范可尼贫血患者的细胞缺乏修复一种毒性很大的DNA损伤类型的能力，即链间交联，其将DNA的沃森和克里克链共价连接在一起，从而阻止它们的分离。这种类型的DNA损伤可能是由环境中的毒素、细胞过程代谢物或癌症治疗期间的化疗引起的。使用国际范可尼贫血登记处的患者的全外显子组测序，我们有 发现了一个关键的细胞重组酶RAD51的突变。患者出生时发育异常，包括拇指缺失。目前骨髓功能正常。患者成纤维细胞对交联剂过敏，但与其他范可尼贫血患者成纤维细胞中观察到的高得多的水平相比，交联剂暴露后的染色体断裂水平仅轻度升高。尽管用一系列DNA损伤剂治疗后RAD51病灶形成减少和延迟，但患者成纤维细胞对电离辐射不敏感。此外，患者成纤维细胞在用DNA交联药物处理后显示野生型水平的姐妹染色单体交换，并且能够进行同源重组以修复功能失调的GFP基因。总的来说，这些结果表明在患者细胞中双链断裂的同源依赖性修复没有受损。进一步的实验表明，RAD51的基本功能，在网站的停滞， 复制叉，很可能是预防肿瘤发生所必需的关键功能是保护肿瘤细胞。 DNA被降解，阻止正确的修复。在拟议的实验中，我们努力了解 RAD51及其结合伴侣在交联修复过程中的功能机制。首先，我们建议使用患者细胞系来了解DNA链间交联修复过程中RAD51和BRCA2的同源重组独立功能。其次，我们想了解RAD 51和范可尼贫血通路之间的相互作用。最后，我们将研究RAD51相互作用蛋白如何影响RAD51在停滞复制叉的功能。总体目标是了解RAD 51和相关因子如何通过在未受干扰的DNA复制期间发挥作用来预防肿瘤发生。