Repair and Processing of DNA Crosslinks

描述(申请人提供)：连接两条DNA链的链间DNA交联物(ICL)毒性很高，因为它们是DNA复制和转录的有效阻碍。这种交联剂是由临床相关的化疗药物和环境来源形成的。人类细胞可以修复其中一些具有挑战性的损伤，但交联链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，并分析其修复和人类酶的处理。目的1是利用独特的底物和新的检测方法来确定特定的核苷酸切除修复(NER)蛋白和反应在补骨脂素ICL修复中的参与。首先，将通过检查在NER中有缺陷的细胞提取物来测试NER酶在交联链两侧切割中的参与程度，并预测NER有缺陷的XP提取物将没有两个切口处。XPC-HR23B似乎是DNA扭曲的初始识别因子。我们将测试纯化的XPC-HR23B复合体是否能与含有单一ICL的DNA结合，并将这种结合与其他特征的扭曲进行比较。在NER中，一个中间的特征步骤是在病变周围形成一个开放的复合体，其中DNA变成短暂的单链。为了测试DNA是否可以在交联物的两侧打开以形成切开前的复合体，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的一条臂，如果不足以释放，将对人类细胞提取物进行分级，以纯化额外的因子。目的2是确定模拟DNA复制分叉或转录位点的Y结构是否为ERCC1-XPF的底物。我们假设这样的Y-结构可以在DNA复制或转录过程中形成，并形成底物，供ERCC1-XPF作用以释放交联剂的一条臂。为了研究这一点，将首先测试在Y结构的每一臂上包含双链DNA的模型DNA复制叉子作为ERCC1-XPF底物的适用性。据预测，当ERCC1-XPF相对于叉子适当的位置时，将能够在ICL的两侧进行切割。带有DNA或RNA聚合酶的模型底物也将被测试。目的3研究POLQ家族DNA聚合酶的生化活性和与DNA修复相关的细胞功能。纯化的POLQ和POLN是我们实验室发现的一种新酶，将测试其绕过DNA损伤的能力，包括脱钩的DNA交联键。这些酶的保真度将被测量。为了研究POLQ和POLN的细胞功能，将进行siRNA对这些酶的抑制，然后测试对DNA交联剂的变化敏感性。将检查DNA损伤后细胞定位的变化。通过免疫沉淀法从转基因细胞中分离POLN和POLQ，并对共纯化蛋白进行分析，以确定参与DNA交联修复的新因素。这些因素可能被证明是未来抑制肿瘤细胞内ICL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.