Direct Analysis of Fork Blockage and DNA Repair in vivo

描述(由申请人提供)：本申请侧重于复制分叉失败和重新启动的途径，特别强调分叉对模板中特定部位病变的反应。需要分析的一种损伤类型涉及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针对的是II型DNA拓扑异构酶。这些抑制剂稳定了切割复合体，这是一种由酶共价连接在酶介导的DNA断裂部位的反应中间产物。裂解复合体是细胞毒性所必需的，但不是充分的，有证据表明，DNA复制对于将裂解复合体转化为细胞毒性和潜在的突变损伤是重要的。我们将试图在抗肿瘤药物作用的噬菌体T4模型系统和喹诺酮类药物治疗的大肠杆菌中破译细胞毒性损伤产生的途径。第二种类型的损伤涉及胞嘧啶甲基酶和DNA之间的共价复合体，但在这种情况下，没有固有的DNA断裂。氮胞嘧啶相关化合物包括抗白血病和其他疾病的抗肿瘤药物，可在甲基酶识别位点诱导共价甲基酶-DNA复合体。 这些化合物具有复杂的作用机制，在这一应用中的实验将集中在蛋白质-DNA复合体形成的后果上。我们将验证甲基酶-DNA加合物阻止复制分叉的假设，并探索这种分叉阻止导致DNA损伤的可能性，这可能与药物的细胞毒性和诱导突变有关。最后，基本部位是一种常见的DNA损伤，发生在正常生长条件下，如果不修复，可能会引发复制分叉堵塞。我们将尝试将具有特定位点的DNA输送到活的大肠杆菌细胞中，并对这些位点的修复和复制分叉阻塞进行物理分析。

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.