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.

项目总结/摘要 细胞暴露于环境因子，如辐射、重金属、空气污染物和 诱变化学品，产生DNA双链断裂（DSB）和其他染色体 损伤，也可能导致复制应激。这种环境诱导的染色体 损伤和应激通过保守的机制-同源重组（HR）来消除。 HR的缺陷及其失调导致基因组不稳定、癌症和其他疾病。 包括解旋酶在内的几种核酸马达蛋白参与了 影响HR结果和DNA损伤修复，减轻细胞复制叉应激， 介导R环的分解。 ZGRF 1是一种核酸马达，与siRNA中的HR和DNA交联修复有关。 基于基因组筛选。我们已经发现，工程化的ZGRF 1-/-细胞对 丝裂霉素C（MMC）治疗和药物治疗后累积染色体畸变。 重要的是，纯化的ZGRF 1显示DNA依赖性ATP酶、D环和R环解离， 复制叉回归活动，表明ZGRF 1的功能直接调节HR和 介导复制叉修复和R环分解。在这个项目中，我们将应用我们的大量 在核酸马达蛋白的分子研究的专业知识，以确定机制， ZGRF 1完成其生物学功能。在目标1中，我们将执行各种遗传和 细胞学研究检查ZGRF 1突变细胞在DNA损伤修复、复制 分叉维护/维修，以及R回路解决方案。我们将确定 ZGRF 1核酸运动活性与我们已经产生的ATP酶缺陷突变体。 在目标2中，我们将定义ZGRF 1的生化属性，并进行协同研究。 免疫沉淀和生化下拉来鉴定这种马达蛋白的相互作用物。在Aim中 3、我们将研究ZGRF 1突变体的生化和遗传缺陷， 指状结构域，蛋白质-蛋白质相互作用受损，或在癌症中发现。结果从我们的 该项目将阐明ZGRF 1在核过程中的作用，这些作用与描述 致病突变和染色体重排是如何在缺乏 核酸马达R环分辨率的结果预计将有助于 开发新的策略，以避免暴露于 环境压力和诱变剂。