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: 9753247
• 负责人: Patrick Sung
• 金额: $ 22.88万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 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 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9753247
• 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 biosynthesis; 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; causal variant; cell transformation; cofactor; environmental agent; helicase; homologous recombination; insight; mutant; novel strategies; prevent; protein protein interaction; repaired; replication stress; 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 细胞暴露于环境因素，如辐射、重金属、空气污染物和 致突变化学物质，产生脱氧核糖核酸双链断裂S等染色体 损害，也可引起复制应激。这种环境诱导的染色体 损伤和压力被一种保守的机制-同源重组(HR)消除。 HR的缺陷及其放松调控会导致基因组不稳定、癌症和其他疾病。 几种核酸马达蛋白，包括解旋酶，已经被牵连到机制中。 影响HR结局和DNA损伤修复，减轻细胞复制分叉压力，以及 调解R-环路的解决。 ZGRF1是一种核酸马达，参与了siRNA的HR和DNA交联修复。 基于基因组的筛选。我们发现工程化的ZGRF1-/-细胞对 丝裂霉素C(MMC)治疗和药物治疗时积累的染色体畸变率。 重要的是，纯化的ZGRF1表现出DNA依赖的ATPase，D-环和R-环的解离，以及 复制分叉回归活动，提示ZGRF1直接调节HR和 协调复制分叉修复和R-循环解决。在这个项目中，我们将运用我们可观的 在核酸马达蛋白的分子研究方面的专门知识，以确定 ZGRF1完成了其生物学功能。在目标1中，我们将执行各种遗传和 检测ZGRF1突变细胞DNA损伤修复、复制缺陷的细胞学研究 叉子维护/维修，以及R-LOOP解决方案。我们将确定它的生物学相关性 ZGRF1与我们产生的ATPase缺陷突变株的核酸马达活性。 在目标2中，我们将定义ZGRF1的生化属性，并进行联合 免疫沉淀和生化下拉以确定该马达蛋白的相互作用。在AIM 3，我们将调查ZGRF1突变体在锌中发生的生化和遗传缺陷 指状域，蛋白质-蛋白质相互作用受损，或在癌症中发现。我们的结果是 该项目将阐明ZGRF1在与描绘密切相关的核过程中的作用 细胞中致病突变和染色体重排是如何发生的 核酸马达。关于R环分辨率的结果预计将有助于 开发避免暴露在空气中的R环积累的新策略 环境压力和诱变剂。