Repair and Processing of DNA Crosslinks

描述（由申请人提供）：连接两条DNA链的链间DNA交联（ICLs）是剧毒的，因为它们是DNA复制和转录的有效阻断。这种交联是由临床相关的化疗药物和环境来源形成的。人类细胞可以修复这些具有挑战性的病变，但交联修复的详细机制尚不清楚。这里提出的研究利用系统将单个ICL放置在DNA中，并分析其在人类酶的修复和处理。目的1是利用独特的底物和新的测定方法来确定特定核苷酸切除修复（NER）蛋白和反应在补骨脂素ICL修复中的作用。首先，将通过检测有NER缺陷的细胞提取物来测试NER酶参与交联两侧的切割，并预测有NER缺陷的XP提取物将缺少两个切口。XPC-HR23B似乎是DNA畸变的初始识别因子。我们将测试纯化的XPC-HR23B复合物是否可以与含有单个ICL的DNA结合，并将这种结合与其他特征扭曲进行比较。在NER中，中间特征步骤是在病变周围形成开放复合物，其中DNA变为短暂的单链。为了测试DNA是否可以在交联的任何一侧打开以形成切口前复合物，将使用化学足迹方法。我们将确定6个核心NER因子XPA， XPC-HR23B等是否足以释放ICL的一个臂，如果不足够，将对人细胞提取物进行分离以纯化其他因子。目的2是确定模拟DNA复制叉或转录位点的Y结构是否为ERCC1-XPF的底物。我们假设这种y型结构可能在DNA复制或转录过程中形成，并形成ERCC1-XPF释放交联臂的底物。为了研究这一点，将首先测试Y结构的每条臂上含有双链DNA的模型DNA复制叉是否适合作为ERCC1-XPF的底物。据预测，ERCC1-XPF将能够在ICL的两侧切割，当适当地定位于分叉时。模型底物与DNA或RNA聚合酶停止附近的交联也将进行测试。目的3是研究POLQ家族DNA聚合酶的生化活性和细胞功能，以及与DNA修复相关的几个特性。纯化的POLQ和POLN，是我们实验室发现的一种新酶，将被测试绕过DNA损伤的能力，包括未连接的DNA交联。酶的保真度将被测量。为了研究POLQ和POLN的细胞功能，将对这些酶进行siRNA抑制，然后进行对DNA交联剂敏感性变化的测试。DNA损伤后细胞定位的变化将被检查。通过免疫沉淀法从转染细胞中分离出POLN和POLQ，分析共纯化蛋白，确定参与DNA交联修复的新因子。这些因子可能被证明是未来抑制肿瘤细胞内ICLs修复的有用靶点。

DESCRIPTION (provided by applicant): Interstrand DNA crosslinks (ICLs) linking the two strands of DNA are highly toxic because they are such efficient blocks to DNA replication and transcription. Such crosslinks are formed by clinically relevant chemotherapeutic agents, and from environmental sources. Human cells can repair some of these challenging lesions, but the detailed mechanisms of crosslink repair are unknown. The research proposed here makes use of systems to place a single ICL within DNA and analyze its repair and processing by human enzymes. Aim 1 is to make use of unique substrates and new assays in order to determine the involvement of specific nucleotide excision repair (NER) proteins and reactions in repair of a psoralen ICL. First, the involvement of NER enzymes in cleavage on either side of a crosslink will be tested by examining cell extracts defective in NER, with the prediction that NER-defective XP extracts will lack both incisions. XPC-HR23B appears to be an initial recognition factor for DNA distortions. We will test whether the purified XPC-HR23B complex can bind to DNA containing a single ICL, and compare this binding to other characterized distortions. In NER, an intermediate characterized step is formation of an open complex around a lesion, where DNA becomes transiently single-stranded. To test whether the DNA can open up on either side of a crosslink to form a preincision complex, chemical footprinting methods will be used. We will determine whether the 6 core NER factors XPA, XPC-HR23B etc. are sufficient for release of one arm of an ICL and if not, human cell extracts will be fractionated in order to purify additional factors. Aim 2 is to determine whether Y structures that model DNA replication forks or sites of transcription are substrates for ERCC1-XPF. We hypothesize that such Y-structures could form during DNA replication or transcription and form substrates for action by ERCC1-XPF to release one arm of a crosslink. To investigate this, model DNA replication forks containing double-stranded DNA on each arm of a Y structure will first be tested for suitability as a substrate for ERCC1-XPF. It is predicted that ERCC1-XPF will be able to cleave on both sides of the ICL, when appropriately positioned with respect to the fork. Model substrates with DNA or RNA polymerases stalled near the crosslink will also be tested. Aim 3 is to investigate the biochemical activities and cellular functions of POLQ family DNA polymerases with respect to several properties relevant to DNA repair. Purified POLQ and POLN, a new enzyme discovered in our laboratory, will be tested for the ability to bypass DNA damage, including an unhooked DNA crosslink. The fidelity of the enzymes will be measured. To investigate cellular functions of POLQ and POLN, siRNA inhibition of the enzymes will be carried out followed by tests for changed sensitivity to DNA crosslinking agents. Changes in cellular localization after DNA damage will be examined. POLN and POLQ will be isolated from transfected cells by immunoprecipitation, and co-purifying proteins will be analyzed to identify new factors involved in DNA crosslink repair. Such factors might prove to be useful targets in future efforts to inhibit repair of ICLs in tumor cells.