Cellular responses to interstrand cross-links in S phase: replication fork

DNA链间交联剂是人类从外源和内源来源暴露的高度有害的损伤和强效诱变剂。此外，这类药物，包括环磷酰胺、顺铂、白消安和丝裂霉素C，被广泛用作抗癌化疗药物。然而，细胞对这些药物在细胞周期和DNA修复方面的反应仍然知之甚少。在上一个资助周期中，我们能够鉴定出一些参与细胞ICL反应的新蛋白质。此外，我们还开发了一些新的检测方法， 探索ICL修复机制的能力。在哺乳动物细胞中似乎存在至少两种可区分的ICL修复途径，其中之一发生在G1/G 0，第二种是由S期期间停滞的复制叉诱导的。这个项目的重点将是增加我们对ICL修复的S期途径的各个方面的理解。具体来说，我们研究招聘的修复和检查点蛋白ICLs使用一种新的激光显微照射的方法，可用于交联pesticen的哺乳动物细胞核的一个定义的子区域。其次，我们将研究分叉崩溃的机制，并定义直接参与ICL去除的蛋白质。第三，我们将研究候选蛋白在ICL修复过程的各个阶段中的作用。第四，我们将分离停滞的复制叉，并使用质谱法鉴定参与修复和检查点功能的蛋白质，这些蛋白质被招募到这些结构中。这些目标的成功实现将大大增加我们对哺乳动物细胞中ICL加工机制的理解， 从而导致潜在的新的或增强的癌症化疗。

DNA interstrand crosslinking agents are highly deleterious lesions and potent mutagens to which humans are exposed from both exogenous and endogenous sources. In addition, this class of drugs, which include cyclophosamide, cis-platin, busulfan, and mitomycin C, are widely used as anti-cancer chemotherapeutics. Nevertheless, the cellular responses to these drugs in terms of both the cell cycle and DNA repair remain poorly understood. During the last grant cycle we were able to identify a number of novel proteins involved in the cellular ICL response. In addition, we have developed a number of new assays that have increased our ability to probe the mechanisms of ICL repair. There appears to be at least two distinguishable pathways of ICL repair in mammalian cells one of which occurs in G1/G0, and a second that is induced by stalled replication forks during S phase. The focus of this project will be on increasing our understanding of the various aspects of the S phase pathway of ICL repair. Specifically, we examine recruitment of repair and checkpoint proteins to ICLs using a novel laser microirradiation approach which can be used to crosslink psoralen to a defined subregion of the mammalian nucleus. Secondly, we will examine the mechanisms of fork collapse, and define proteins that are directly involved in ICL removal. Thirdly, we will investigate the role of candidate proteins in various stages of ICL repair processing. Fourthly, we will isolate stalled replication forks and using mass spectrometry we will identify proteins involved in repair and checkpoint functions that are recruited to these structures. Together the successful completion of these aims should greatly increase our understanding of the mechanisms by which ICLs are processed in mammalian cells, and thereby lead to potential new or enhanced chemotherapies for cancer.