Direct Analysis of Fork Blockage and DNA Repair in vivo

体内叉阻断和 DNA 修复的直接分析

• 批准号: 6828465
• 负责人: KENNETH N KREUZER
• 金额: $ 27.72万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6828465
• 关键词: DNA damage; DNA repair; DNA replication origin; DNA topoisomerases; Escherichia coli; adduct; aminoacridines; bacteriophage T4; cytotoxicity; enzyme inhibitors; molecular genetics; oligonucleotides; quinoline

DESCRIPTION (provided by applicant): This application focuses on the pathways of replication fork failure and restart, with a particular emphasis on the response of the fork to site-specific lesions in the template. One type of lesion to be analyzed involves inhibitors of DNA topoisomerases. Several important anticancer drugs, as well as the antibacterial quinolones target type II DNA topoisomerases. These inhibitors stabilize the cleavage complex, a reaction intermediate consisting of the enzyme covalently attached at sites of enzyme-mediated DNA breakage. The cleavage complex is necessary but not sufficient for cytotoxicity, and evidence indicates that DNA replication is important for converting cleavage complexes into cytotoxic and potentially mutagenic lesions. We will attempt to decipher the pathway of cytotoxic lesion generation, in both a phage T4 model system for antitumor drug action and in quinolone-treated Escherichia coli. A second type of lesion involves covalent complexes between cytosine methylases and DNA, but in this case, there is no inherent DNA break. Covalent methylase-DNA complexes are induced at the methylase recognition sites by treatment of cells with aza-cytosine-related compounds, which include antitumor agents active against leukemias and other diseases. These compounds have complex mechanisms of action, and the experiments in this application will focus on the consequences of protein-DNA complex formation. We will test the hypothesis that methylase-DNA adducts block the replication fork, and explore the possibility that this fork blockage leads to DNA damage that might be involved in drug cytotoxicity and induced mutagenesis. Finally, abasic sites are a common DNA lesion that occurs under normal growth conditions, and if unrepaired, likely trigger replication fork blockage. We will attempt to deliver DNA with site-specific abasic sites into living E. coli cells, and physically analyze repair and replication fork blockage at these sites.

描述（由申请人提供）：本申请重点关注复制叉失败和重新启动的途径，特别强调复制叉对模板中特定位点损伤的响应。待分析的一类损伤涉及 DNA 拓扑异构酶抑制剂。几种重要的抗癌药物以及抗菌喹诺酮类药物都以 II 型 DNA 拓扑异构酶为靶标。这些抑制剂可稳定裂解复合物，这是一种反应中间体，由共价连接在酶介导的 DNA 断裂位点的酶组成。裂解复合物对于细胞毒性来说是必要的，但还不够，证据表明 DNA 复制对于将裂解复合物转化为细胞毒性和潜在的诱变损伤很重要。我们将尝试破译抗肿瘤药物作用的噬菌体 T4 模型系统和喹诺酮处理的大肠杆菌中细胞毒性病变产生的途径。第二种类型的损伤涉及胞嘧啶甲基化酶和 DNA 之间的共价复合物，但在这种情况下，不存在固有的 DNA 断裂。通过用氮杂胞嘧啶相关化合物（包括有效对抗白血病和其他疾病的抗肿瘤药物）处理细胞，在甲基化酶识别位点诱导共价甲基化酶-DNA 复合物。 这些化合物具有复杂的作用机制，本申请中的实验将重点关注蛋白质-DNA 复合物形成的后果。我们将检验甲基化酶-DNA 加合物阻断复制叉的假设，并探讨这种复制叉阻断导致 DNA 损伤的可能性，而 DNA 损伤可能与药物细胞毒性和诱导突变有关。最后，脱碱基位点是正常生长条件下发生的常见 DNA 损伤，如果不修复，可能会引发复制叉阻塞。我们将尝试将具有位点特异性脱碱基位点的 DNA 传递到活的大肠杆菌细胞中，并对这些位点的修复和复制叉阻塞进行物理分析。