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

S 期细胞对链间交联的反应：复制叉

• 批准号: 8211103
• 负责人: RANDY J LEGERSKI
• 金额: $ 71.52万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-21 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8211103
• 关键词: Anatomy; Biological Assay; Busulfan; Bypass; Cell Cycle; Cell Cycle Checkpoint; Cell Nucleus; Cells; Chemotherapy-Oncologic Procedure; Complex; DNA; DNA Damage; DNA Interstrand Crosslinking; DNA Repair; Digoxigenin; ERCC1 gene; Excision; Ficusin; G0 Phase; Grant; Health; Hela Cells; Human; Kinetics; Knowledge; Lasers; Lead; Lesion; MLH1 gene; MSH2 gene; MSH3 gene; MSH6 gene; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Mediating; Minor; Mismatch Repair; Mitomycins; Mitotic; Modeling; Mutagens; Nucleotide Excision Repair; Pathway interactions; Pharmaceutical Preparations; Phase; Process; Proteins; Psoralens; Reaction; Recruitment Activity; Role; S Phase; Site; Source; Staging; Structure; Technology; adduct; cancer therapy; crosslink; homologous recombination; improved; mammalian genome; new technology; new therapeutic target; novel; programs; repaired; research study; response; restoration

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.

DNA链间交联药是高度有害的病变和有效的诱变剂，从外源和内源性来源暴露于人类。此外，这类药物（包括环磷酸，顺铂，布尔素和丝裂霉素C）被广泛用作抗癌化学治疗药。然而，从细胞周期和DNA修复方面，细胞对这些药物的反应仍然很少。在最后一个赠款周期中，我们能够鉴定出许多参与细胞ICL反应的新型蛋白质。此外，我们开发了许多新的测定法 能够探测ICL修复机制的能力。在哺乳动物细胞中，ICL修复的至少有两种可区分的途径，其中一种发生在G1/G0中，第二个是由S相中的停滞复制叉引起的。该项目的重点将是增加我们对ICL修复S相途径各个方面的理解。具体而言，我们使用新型的激光微辐照方法检查了修复和检查点蛋白至ICL的募集，该方法可用于交叉链接牛皮利亚与哺乳动物核的确定子区域。其次，我们将检查叉子塌陷的机制，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修复处理的各个阶段的作用。第四，我们将隔离停滞的复制叉，并使用质谱法，我们将确定参与修复和检查点功能的蛋白质。共同完成这些目标的成功应大大增加我们对哺乳动物细胞中ICL进行处理的机制的理解， 从而导致潜在的新化学疗法癌。