Regulation of DnaA and replication initiation in Bacillus subtilis

描述(申请人提供)：复制在所有生物体中都是一个保守且必不可少的过程，复制组件是潜在的抗生素靶点，复制的错误调控可以促进多细胞生物体的肿瘤发生。这项应用研究细菌复制起始，这是一个简化的模型，因为存在具有单一复制起点ORIC的单一染色体和单一复制起始蛋白DNAA。DNAA与ORIC结合并指导复制机器的组装。DNAA与核苷酸的结合调节DNAA的活性，但由于DNAA的重要性、DNAA的自动调节以及基于ORIC的质粒复制模型不能忠实地复制染色体复制，分析这种调节被证明是困难的。以枯草芽孢杆菌为模型，本应用将：1)确定DNAA的核苷酸结合在调节DNAA-ORIC相互作用中的作用；2)在体内定位枯草杆菌与DNAA之间的相互作用；3)剖析YABA和夹子对DNAA的调节。为了实现这些目标，我将使用定点突变来产生锁定在核苷酸空、DNAA-ATP或DNAA-ADP形式中的DNAA构建体。我将使用这些构建物来探索核苷酸结合/水解在调节枯草杆菌DNAA-ORIC相互作用和开放复合体形成中的作用。利用染色质免疫沉淀和体内化学足迹，将直接在细菌染色体上探测DNAA与复制的染色体起源的相互作用。通过使用一株可以从ORIC或异源、Dna A非依赖性复制起点Orin复制的枯草杆菌菌株，I将能够表征Dna A中其他致命突变对体内ORIC结合的影响，从而确定核苷酸结合在支持体内复制启动中的作用。最后，我将通过研究Yaba和夹子在调节枯草杆菌Dna A的核苷酸结合/水解、Dna A-Oric相互作用和Dna A介导的复制启动中所起的作用，剖析在枯草杆菌中存在的Rida类机制。与公共卫生相关：这项应用描述了导致细菌基因组复制的事件。由于复制细胞遗传物质的机制是抗生素的潜在靶点，而且基因组复制的错误调控可能会促进高等生物体中癌症的形成，这项工作将有助于定义抗生素靶点，并加深我们对可能促进癌症的分子事件的理解。

DESCRIPTION (provided by applicant): Replication is a well-conserved and essential process in all organisms, replication components are potential antibiotic targets and misregulation of replication can promote oncogenesis in multicellular organisms. This application examines bacterial replication initiation, which serves as a simplified model due to the presence of a single chromosome with a single replication origin, oriC, and a single replication initiation protein, DnaA. DnaA binds to oriC and directs the assembly of the replication machinery. Nucleotide binding by DnaA regulates DnaA activity, but dissecting this regulation has proven difficult due to the essentiality of DnaA, the autoregulation of DnaA and the failure of oriC-based plasmid replication models to faithfully reproduce chromosomal replication. Using the bacterium Bacillus subtilis as a model, this application will: 1) determine the role of DnaA's nucleotide binding in regulating DnaA-oriC interaction, 2) map the interactions between B. subtilis oriC and DnaA in vivo, and 3) dissect the regulation of DnaA by YabA and the ¿-clamp. To accomplish these aims, I will use site-directed mutagenesis to generate DnaA constructs that are locked into the nucleotide-empty, DnaA-ATP or DnaA-ADP forms. I will use these constructs to probe the role of nucleotide binding/hydrolysis in regulating B. subtilis DnaA-oriC interaction and open complex formation. DnaA's interaction with the chromosomal origin of replication will be probed directly on the bacterial chromosome using chromatin immunoprecipitation and in vivo chemical footprinting. By using a B. subtilis strain that can replicate from either oriC or a heterologous, DnaA-independent origin of replication, oriN, I will be able to characterize the effect of otherwise lethal mutations in DnaA on oriC binding in vivo, and thereby define the role of nucleotide binding in supporting replication initiation in vivo. Last, I will dissect the RIDA-like mechanism proposed to exist in B. subtilis by characterizing the role YabA and the ¿-clamp play in regulating B. subtilis DnaA's nucleotide binding/hydrolysis, DnaA-oriC interaction and DnaA-mediated replication initiation. PUBLIC HEALTH RELEVANCE: This application characterizes the events that lead to genome duplication in bacteria. Because the machinery that duplicates a cell's genetic material is a potential target for antibiotics, and because misregulation of genome duplication can promote cancer formation in higher organisms, this work will help to define antibiotic targets and refine our understanding of the molecular events that can promote cancer.