Roles of the nucleic acid motor protein ZGRF1 in chromosome damage repair

核酸马达蛋白ZGRF1在染色体损伤修复中的作用

• 批准号: 9575041
• 负责人: Patrick Sung
• 金额: $ 11.07万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-15 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9575041
• 关键词: ATP phosphohydrolase; Affect; Air Pollutants; BARD1 gene; BRCA1 gene; BRCA2 gene; Biochemical; Biochemical Genetics; Biological; Biological Process; Cancer Etiology; Cells; Chemicals; Chromosome abnormality; Chromosomes; Co-Immunoprecipitations; Cytology; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA crosslink; DNA replication fork; DNA-dependent ATPase; Defect; Development; Disease; Dissociation; Engineering; Exposure to; Genetic; Genetic Transcription; Genome; Genomics; Goals; Heavy Metals; Human; Hypersensitivity; Impairment; Lead; Length; Lesion; Light; Maintenance; Malignant Neoplasms; Mediating; Mitomycins; Molecular; Motor; Motor Activity; Mutagens; Mutation; Neurodegenerative Disorders; Nuclear; Nucleic Acids; Null Lymphocytes; Outcome; Pathogenicity; Pathway interactions; Pharmacotherapy; Play; Process; Proteins; RNA Helicase; Radiation; Regulation; Resistance; Resolution; Role; Small Interfering RNA; Stress; Zinc Fingers; base; cell transformation; cofactor; disease-causing mutation; environmental agent; helicase; homologous recombination; insight; mutant; novel strategies; prevent; protein protein interaction; repaired; response; stem; telomere; tool

PROJECT SUMMARY/ABSTRACT Exposure of cells to environmental agents, such as radiation, heavy metals, air pollutants and mutagenic chemicals, generates DNA double-strand breaks (DSB)s and other chromosomal lesions, and can also cause replicative stress. Such environmentally induced chromosomal lesions and stress are eliminated by a conserved mechanism - homologous recombination (HR). Defects in HR and its deregulation lead to genome destabilization, cancer, and other diseases. Several nucleic acid motor proteins, including helicases, have been implicated in mechanisms that affect HR outcome and DNA damage repair, relieve cells from replication fork stress, and mediate the resolution of R-loops. ZGRF1, a nucleic acid motor, has been implicated in HR and DNA crosslink repair in siRNA- based genomic screens. We have found that engineered ZGRF1-/- cells are hypersensitivity to mitomycin C (MMC) treatment and accumulate chromosome aberrations upon drug treatment. Importantly, purified ZGRF1 shows DNA dependent ATPase, D-loop and R-loop dissociation, and replication fork regression activities, suggesting that ZGRF1 functions directly to regulate HR and mediate replication fork repair and R-loop resolution. In this project, we will apply our considerable expertise in molecular studies of nucleic acid motor proteins to define the mechanisms by which ZGRF1 accomplishes its biological functions. In Aim 1, we will perform a variety of genetic and cytological studies to examine ZGRF1 mutant cells for defects in DNA damage repair, replication fork maintenance/repair, and also R-loop resolution. We will ascertain the biological relevance of the ZGRF1 nucleic acid motor activity with an ATPase defective mutant that we have generated. In Aim 2, we will define the biochemical attributes of ZGRF1, and carry out co- immunoprecipitation and biochemical pulldown to identify interactors of this motor protein. In Aim 3, we will investigate the biochemical and genetic defects of ZGRF1 mutants altered in the zinc finger domain, impaired for protein-protein interactions, or found in cancer. The results from our project will shed light on the roles of ZGRF1 in nuclear processes that are germane for delineating how disease causative mutations and chromosome rearrangements arise in cells deficient in nucleic acid motors. The results on R-loop resolution are expected to contribute toward the development of novel strategies to avoid the accumulation of R-loops upon exposure to environmental stress and mutagens.

项目摘要/摘要 将细胞暴露于环境药物，例如辐射，重金属，空气污染物和 诱变化学物质，产生DNA双链断裂（DSB）和其他染色体 病变，也可能引起复制压力。这种环境诱导的染色体 病变和应力通过保守的机制 - 同源重组（HR）消除。 人力资源缺陷及其放松管制导致基因组不稳定，癌症和其他疾病。 几种核酸运动蛋白（包括解旋酶）已与机理有关 这会影响人力资源结果和DNA损伤修复，缓解细胞免受复制叉应力，以及 调解R环的分辨率。 ZGRF1是一种核酸运动，已与siRNA-中的HR和DNA交联修复有关 基因组筛选。我们发现工程化的ZGRF1 - / - 细胞是超敏反应的 丝裂霉素C（MMC）治疗并在药物治疗后积累染色体畸变。 重要的是，纯化的ZGRF1显示DNA依赖性ATPase，D-Loop和R-loop解离，以及 复制叉回归活动，表明ZGRF1直接起作用以调节人力资源和 介导复制叉修复和R环分辨率。在这个项目中，我们将应用我们的大量 核酸运动蛋白分子研究的专业知识，以定义该机制 ZGRF1完成了其生物学功能。在AIM 1中，我们将执行各种遗传和 细胞学研究检查ZGRF1突变细胞是否有DNA损伤修复的缺陷，复制 分叉维护/维修以及R-loop分辨率。我们将确定 我们已经产生的ATPase有缺陷突变体的ZGRF1核酸运动活性。 在AIM 2中，我们将定义ZGRF1的生化属性，并进行共同 免疫沉淀和生化下拉，以鉴定该运动蛋白的相互作用者。目标 3，我们将研究锌中改变的ZGRF1突变体的生化和遗传缺陷 手指结构域，因蛋白质蛋白质相互作用而受损或在癌症中发现。我们的结果 项目将阐明ZGRF1在核过程中的作用 疾病的病因突变和染色体重排如何在缺乏的细胞中出现 核酸电动机。 R环分辨率的结果有望有助于 开发新型策略，以避免暴露于R-loop的积累 环境压力和诱变剂。