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 当蛋白与DNA形式结合时，DNA蛋白交联（DPC）形成 由于正常的细胞过程（例如脂质过氧化，组蛋白脱甲基化，DNA）赞助 复制，转录和DNA修复。 DPC可以通过暴露于抗肿瘤药物，过渡来诱导 金属，紫外线和γ辐射。 DPC干扰了许多生物学过程，并与 在RUIJS-AALFS综合征患者中观察到的加速衰老和癌症发病率增加。目标的目标 应用是沿基因组绘制DPC病变，研究人类细胞如何识别和去除 这些非常庞大的DPC病变，并确定它们引起诱变性和 细胞死亡。我们的中心假设是未修复的DPC损害了DNA的效率和准确性 复制并有助于上述药物引起的毒性和诱变。我们的研究 计划侧重于三个目标。首先，将使用下一代测序与亲和力下降 和蛋白质精度，以识别易受赞助和异种生物的特定基因组区域 在人类细胞中诱导的DPC形成。其次，我们将阐明蛋白水解处理在DPC中的作用 维修。亲和力捕获，无偏见的搜索和基于候选基因的方法斜纹twill用于识别 DPC的蛋白水解处理和修复所需的蛋白质，确定细胞如何将DPC转换为较小 从基因组中去除DPC所需的肽病变（DPC）和身份临界DNA修复蛋白。 第三，我们将研究DPC和DPC对DNA复制的影响。我们使用DNA POL的体外研究 η表明，过去肽DPC的translesion合成的效率和保真度强烈依赖于DNA 序列上下文。我们将研究序列上下文对旁路效率和诱变性的影响 人类细胞。上下文对旁路效率和保真度的影响的结构基础将研究 通过分子建模和NMR研究。我们将使用新开发的测定法案，员工Pigybac DPC或DPC的转置含有DNA，以检查这些病变对染色体的影响 复制。这些研究将首次检查结构定义的生物学结果 人类细胞中的染色体DPC。