Direct Analysis of Fork Blockage and DNA Repair in vivo

描述（由申请人提供）：该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响应。要分析的一种病变涉及DNA拓扑异构酶抑制剂。一些重要的抗癌药物，以及抗菌喹诺酮类药物靶向II型DNA拓扑异构酶。这些抑制剂稳定了裂解复合物，这是一种反应中间体，由酶共价附着在酶介导的DNA断裂位点上。切割复合体对细胞毒性是必要的，但不是充分的，有证据表明DNA复制对于将切割复合体转化为细胞毒性和潜在的致突变损伤是重要的。我们将尝试在抗肿瘤药物作用的T4噬菌体模型系统和喹诺酮治疗的大肠杆菌中破译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，没有固有的DNA断裂。共价甲基化酶- dna复合物是通过偶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）处理细胞在甲基化酶识别位点诱导的。

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.