DNA Protein Cross-Links:Cellular Effects and Repair Mechanisms

DNA 蛋白质交联：细胞效应和修复机制

• 批准号: 10017995
• 负责人: COLIN R CAMPBELL
• 金额: $ 54.12万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-21 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10017995
• 关键词: Affect; Affinity; Aging; Antineoplastic Agents; Biological; Biological Assay; Biological Process; Bypass; Candidate Disease Gene; Cell Death; Cell Survival; Cell physiology; Cells; Chemicals; Chromatin; Cisplatin; Cleaved cell; DNA; DNA Repair; DNA Repair Gene; DNA Sequence; DNA biosynthesis; DNA lesion; DNA replication fork; DNA-Protein Interaction; DNA-protein crosslink; Disease; Embryo; Environmental Pollutants; Excision; Exposure to; Failure; Funding; Gamma Rays; Genetic; Genetic Recombination; Genetic Transcription; Genome; Genomic Instability; Genomic Segment; Germ-Line Mutation; Goals; Histones; Human; In Vitro; Incidence; Ionizing radiation; Lesion; Link; Lipid Peroxidation; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mechlorethamine; Metabolism; Methods; Molecular; Mus; Nucleotide Excision Repair; Occupational; Outcome; Pathology; Pathway interactions; Patients; Peptide Hydrolases; Peptides; Precipitation; Primary carcinoma of the liver cells; Process; Progeria; Proteins; Proteolytic Processing; Proteomics; Rad30 protein; Reactive Oxygen Species; Research; Role; Site; Structure; Syndrome; System; Time; Toxic Environmental Substances; Toxic effect; Transition Elements; Ultraviolet Rays; Xenobiotics; Xenopus; adduct; adverse outcome; age related; antitumor drug; base; chromosome replication; crosslink; demethylation; early onset; environmental agent; erythritol anhydride; experimental study; genome integrity; genotoxicity; human DNA; molecular modeling; next generation sequencing; protease E; recruit; repaired; tumorigenesis

Project Summary DNA-protein cross-links (DPCs) are formed when proteins become covalently bound to DNA form spontaneously as a result of normal cellular processes such as lipid peroxidation, histone demethylation, DNA replication, transcription, and DNA repair. DPCs can be induced by exposure to anti-tumor drugs, transition metals, UV light, and γ-radiation. DPCs interfere with many biological processes and are implicated in the accelerated aging and increased cancer incidence observed in Ruijs-Aalfs syndrome patients. The goal of this application is to map DPC lesions along the genome, investigate how human cells recognize and remove these exceedingly bulky DPC lesions, and to identify the mechanisms by which they cause mutagenicity and cell death. Our central hypothesis is that unrepaired DPCs compromise the efficiency and accuracy of DNA replication and contribute to the toxicity and mutagenicity induced by the agents listed above. Our research plan focuses on three aims. First, will use next generation sequencing in combination with affinity pull down and protein precipitation to identify specific genomic regions susceptible to spontaneous and xenobiotic- induced DPC formation in human cells. Second, we will elucidate the role of proteolytic processing in DPC repair. Affinity capture, unbiased searches, and candidate gene-based approaches twill be used to identify proteins required for proteolytic processing and repair of DPCs, determine how cells convert DPCs to smaller peptide lesions (DpCs), and identity critical DNA repair proteins required for DPC removal from the genome. Third, we will investigate the effects of DPCs and DpCs on DNA replication. Our in vitro studies using DNA Pol η showed that efficiency and fidelity of translesion synthesis past peptide DpCs is strongly dependent on DNA sequence context. We will examine the effects of sequence context on bypass efficiency and mutagenicity in human cells. The structural basis for the context effects on the efficiency and fidelity of bypass will be studied by molecular modeling and NMR studies. We will use a newly developed assay that employs pigyBac transposition of DpC or DPC containing DNA to examine the effects these lesions have on chromosome replication. These studies will for the first time examine the biological outcomes of structurally defined chromosomal DPCs in human cells.

项目摘要 DNA-蛋白质交联（DPC）是蛋白质与DNA共价结合形成的。 作为正常细胞过程的结果，如脂质过氧化、组蛋白去甲基化、DNA 复制、转录和DNA修复。DPC可以通过暴露于抗肿瘤药物、过渡 金属、紫外线和γ辐射。DPC干扰许多生物学过程，并参与 在Ruijs-Aalfs综合征患者中观察到加速衰老和增加癌症发病率。这个目标 其应用是沿着基因组绘制DPC病变沿着，研究人类细胞如何识别和去除 这些非常庞大的DPC病变，并确定其引起致突变性的机制， 细胞死亡我们的中心假设是，未修复的DPC损害DNA的效率和准确性 复制，并有助于上述试剂诱导的毒性和致突变性。我们的研究 计划有三个目标。首先，将使用下一代测序结合亲和下拉 和蛋白质沉淀来识别易受自发性和外源性生物质影响的特定基因组区域- 在人细胞中诱导DPC形成。其次，我们将阐明蛋白水解加工在DPC中的作用 修复.亲和捕获、无偏搜索和基于候选基因的方法可用于识别 蛋白水解加工和修复DPC所需的蛋白质，决定细胞如何将DPC转化为更小的 肽损伤（DpCs），以及从基因组中去除DPC所需的身份关键DNA修复蛋白。 第三，我们将研究DPC和DpCs对DNA复制的影响。我们的体外研究使用DNA Pol η表明跨损伤合成肽DpCs的效率和保真度强烈依赖于DNA 序列上下文。我们将研究序列背景对旁路效率和致突变性的影响， 人类细胞。将研究上下文对旁路效率和保真度影响的结构基础 通过分子建模和核磁共振研究。我们将使用一种新开发的检测方法， 含有DNA的DpC或DPC的转座，以检查这些病变对染色体的影响 复制的这些研究将第一次检查结构定义的生物学结果。 人类细胞中的染色体DPC。