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

DNA 链间交联剂是高度有害的损伤和强效诱变剂，人类可以从外源和内源接触到这些物质。此外，此类药物包括环磷酰胺、顺铂、白消安和丝裂霉素C，广泛用作抗癌化疗药物。然而，我们对这些药物在细胞周期和 DNA 修复方面的细胞反应仍知之甚少。在上一个资助周期中，我们能够鉴定出许多参与细胞 ICL 反应的新型蛋白质。此外，我们还开发了许多新的检测方法，提高了我们的检测能力。 探索 ICL 修复机制的能力。哺乳动物细胞中似乎至少存在两种​​可区分的 ICL 修复途径，其中一种发生在 G1/G0 期，第二种是由 S 期停滞的复制叉诱导的。该项目的重点是增进我们对 ICL 修复 S 期途径各个方面的了解。具体来说，我们使用一种新型激光微辐照方法检查 ICL 中修复和检查点蛋白的募集情况，该方法可用于将补骨脂素交联到哺乳动物细胞核的特定子区域。其次，我们将研究叉塌陷的机制，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 修复过程各个阶段的作用。第四，我们将分离停滞的复制叉，并使用质谱法，我们将识别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。这些目标的成功完成将大大增加我们对 ICL 在哺乳动物细胞中加工机制的理解， 从而带来潜在的新的或增强的癌症化疗方法。

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.