Regulation of DnaA and replication initiation in Bacillus subtilis

描述（由申请人提供）：复制在所有生物中都是一个非常保守和必要的过程，复制成分是潜在的抗生素靶点，复制的错误调节可以促进多细胞生物的肿瘤发生。这个应用程序检查了细菌的复制起始，它作为一个简化的模型，因为存在一条具有单一复制起源的染色体，oriC，和一个单一的复制起始蛋白，dna。dna与oriC结合并指导复制机制的组装。dna的核苷酸结合调节了dna的活性，但由于dna的重要性、dna的自动调节以及基于orc的质粒复制模型无法忠实地复制染色体复制，解剖这种调节已被证明是困难的。本应用程序以枯草芽孢杆菌为模型，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)绘制枯草芽孢杆菌oriC与DnaA在体内的相互作用图谱，3)剖析YabA和¿-clamp对DnaA的调节作用。为了实现这些目标，我将使用定点诱变来产生锁定在核苷酸空，dna - atp或dna - adp形式的dna结构。我将使用这些构建体来探索核苷酸结合/水解在调节枯草芽孢杆菌dna - oric相互作用和开放复合物形成中的作用。dna与染色体复制起源的相互作用将通过染色质免疫沉淀和体内化学足迹直接在细菌染色体上进行探测。通过使用枯草芽孢杆菌菌株，既可以从oriC复制，也可以从异源、不依赖dna的复制源oriN复制，我将能够描述dna中致命突变对体内oriC结合的影响，从而确定核苷酸结合在支持体内复制起始中的作用。最后，我将通过表征YabA和¿-clamp在调节枯草芽孢杆菌dna核苷酸结合/水解、dna - oric相互作用和dna介导的复制起始中的作用，剖析枯草芽孢杆菌中存在的rida样机制。公共卫生相关性：该应用描述了导致细菌基因组复制的事件。因为复制细胞遗传物质的机制是抗生素的潜在靶标，并且因为基因组复制的错误调节可以促进高等生物中癌症的形成，这项工作将有助于确定抗生素的靶标，并完善我们对可以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.