DNA Protein Cross-Links:Cellular Effects and Repair Mechanisms

项目摘要 DNA-蛋白质交联链(DPC)是当蛋白质与DNA形成共价结合时形成的 自发地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可通过接触抗肿瘤药物而诱导，过渡 金属、紫外线和γ辐射。DPC干扰许多生物过程，并与 在Ruijs-Aalfs综合征患者中观察到衰老加速和癌症发病率增加。这样做的目的是 应用是沿着基因组绘制DPC损伤图，研究人类细胞如何识别和移除 这些异常巨大的DPC损伤，并确定它们导致突变和 细胞死亡。我们的中心假设是，未修复的DPC会损害DNA的效率和准确性 复制并导致上述制剂的毒性和致突变性。我们的研究 该计划着眼于三个目标。首先，将使用下一代测序与亲和力下拉相结合 和蛋白质沉淀以确定特定的基因组区域对自发性和外源生物敏感- 在人细胞中诱导形成DPC。第二，我们将阐明蛋白质降解处理在DPC中的作用 修理。亲和力捕获、无偏见搜索和基于候选基因的方法将用于识别 蛋白质降解处理和修复DPC所需的蛋白质，决定了细胞如何将DPC转化为更小的DPC 多肽损伤(DPC)，以及从基因组中移除DPC所需的识别关键DNA修复蛋白。 第三，我们将研究DPC和DPC对DNA复制的影响。我们用DNA Pol进行的体外研究 η表明，跨损伤合成多肽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的影响的结构基础 通过分子模拟和核磁共振研究。我们将使用一种新开发的检测方法 转座DPC或含有DNA的DPC以检测这些损伤对染色体的影响 复制。这些研究将首次检验在结构上定义的 人类细胞中的染色体DPC。

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.