Recognition and processing of complex lesions by components from multiple DNA

DNA链间交联(ICL)是DNA代谢过程中的一大障碍，必须进行修复才能保证细胞的生存。虽然已经做了很多工作来确定细菌和酵母中ICL修复的机制，但它们在哺乳动物细胞中的处理还没有明确的定义。在之前的资助期间，我们在哺乳动物细胞中展示了一种核苷酸切除修复(NER)依赖的、容易出错的三链导向补骨脂素ICL的修复。我们还发现错配修复(MMR)蛋白在无错误的过程中参与了补骨脂素ICL的反应和修复。我们在此续订申请中的工作假设是 NER和错配修复(MMR)蛋白在ICL的识别和初始处理中相互作用，而MMR蛋白独立于NER因子参与ICL诱导的细胞周期调节和细胞凋亡。这项拟议研究的长期目标是阐明从哺乳动物基因组中移除DNA ICL的分子机制，识别在此过程中HR、MMR和NER通路的蛋白质之间的相互作用，并确定这些蛋白质在其他细胞对这些损伤的反应中的作用。具体地说，我们建议：1)检验来自NER和NER的蛋白质的假设 MMR通路在ICL的识别和初始处理过程中相互作用；2)测试MLH1在细胞检查点和DNA ICL的凋亡反应中发挥作用的假设；3)将DNA ICL靶向突变哺乳动物细胞系中的特定基因组位置，以确定DNA修复和重组基因产物在ICL处理中的作用；以及4)评估靶向ICL作为抗增殖治疗剂的潜力。这些研究的结果将为开发利用ICL诱导剂控制人类癌症的改进的靶向策略提供有价值的信息。

DNA interstrand crosslinks (ICLs) present a formidable block to DNA metabolic processes and must be repaired for cell survival. While much work has been done to define the mechanisms of ICL repair in bacteria and yeast, their processing in mammalian cells is not clearly defined. In the previous funding period, we demonstrated an nucleotide excision repair (NER)-dependent, error-prone repair of triplex-directed psoralen ICLs in mammalian cells. We also found that mismatch repair (MMR) proteins are involved in the response to and repair of psoralen ICLs in an error-free process. Our working hypothesis in this renewal application is that NER and mismatch repair (MMR) proteins interact in the recognition and initial processing of ICLs, while MMR proteins, independently of NER factors, are involved in ICL-induced cell-cycle regulation and apoptosis. The long-term objectives of the proposed research are to elucidate molecular mechanisms involved in the removal of DNA ICLs from the mammalian genome, to identify interactions among proteins from HR, MMR, and NER pathways in doing so, and to determine the roles of these proteins in other cellular responses to these lesions. Specifically we propose to: 1) test the hypothesis that proteins from the NER and MMR pathways interact during recognition and initial processing of ICLs; 2) test the proposal that MLH1 functions in cellular checkpoint and apoptosis responses to DNA ICLs; 3) target DNA ICLs to specific genomic sites in mutant mammalian cell lines to determine roles for DNA repair and recombination gene products in processing ICLs; and 4) assess the potential of targeted ICLs as antiproliferative therapeutic agents. The results obtained from these studies will provide valuable information to develop improved targeted strategies to control human cancers using ICL-inducing agents.