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

DNA链间交联剂是人类暴露于外源性和内源性来源的高度有害的病变和强效诱变剂。此外，这类药物包括环磷酰胺、顺铂、丁硫丹和丝裂霉素C，被广泛用作抗癌化疗药物。然而，就细胞周期和DNA修复而言，细胞对这些药物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，我们开发了一些新的分析方法，增加了我们的

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.