Regulation of DnaA and replication initiation in Bacillus subtilis

枯草芽孢杆菌中 DnaA 和复制起始的调节

• 批准号: 7615849
• 负责人: Richard Bradley Weart
• 金额: $ 4.72万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7615849
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Address; Alleles; Animal Model; Antibiotics; Bacillus subtilis; Bacteria; Bacterial Chromosomes; Binding; Biological Assay; Cell Cycle; Cells; Chemicals; Chromosomes; Complex; Cowpox; DNA-Binding Proteins; Engineering; Ensure; Escherichia coli; Event; Failure; Family; Genetic Materials; Genome; Homeostasis; Hydrolysis; In Vitro; Lead; Malignant Neoplasms; Maps; Mechanics; Mediating; Modeling; Molecular; Mutation; Nucleotides; Organism; Plasmids; Play; Polymerase; Process; Proteins; Regulation; Replication Initiation; Replication Origin; Role; Scrapie; Series; Site-Directed Mutagenesis; System; Time; Work; base; chromatin immunoprecipitation; chromosome replication; in vivo; mutant; origin recognition complex; public health relevance; tumorigenesis

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.

描述（由申请人提供）：复制是所有生物体中保守且重要的过程，复制成分是潜在的抗生素靶标，复制的错误调节可以促进多细胞生物体中的肿瘤发生，该应用检查细菌复制起始，作为简化模型。由于存在具有单个复制起点 oriC 和单个复制起始蛋白的单个染色体，DnaA 与 oriC 结合并通过核苷酸结合指导组装。 DnaA 调节 DnaA 活性，但由于 DnaA 的重要性、DnaA 的自动调节以及基于 oriC 的质粒复制模型无法忠实地再现染色体复制，该应用以枯草芽孢杆菌为模型，剖析这种调节已被证明是困难的。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) 绘制枯草芽孢杆菌之间的相互作用图体内 oriC 和 DnaA，以及 3) 剖析 YabA 和 ¿ 对 DnaA 的调节为了实现这些目标，我将使用定点诱变来生成锁定为空核苷酸、DnaA-ATP 或 DnaA-ADP 形式的 DnaA 构建体，我将使用这些构建体来探测核苷酸结合/的作用。将利用细菌染色体直接探测调节枯草芽孢杆菌 DnaA-oriC 相互作用的水解以及开放复合物形成 DnaA 与染色体复制起点的相互作用。通过使用可以从 oriC 或异源、不依赖 DnaA 的复制起点 oriN 复制的枯草芽孢杆菌菌株，我将能够表征 DnaA 中其他致命突变对 oriC 的影响。最后，我将通过表征枯草芽孢杆菌中提出的 RIDA 样机制来剖析。角色 YabA 和 ¿ -clamp 在调节枯草芽孢杆菌 DnaA 的核苷酸结合/水解、DnaA-oriC 相互作用和 DnaA 介导的复制启动中发挥作用。 公共健康相关性：该应用描述了导致细菌基因组复制的事件，因为复制细胞的遗传机制。材料是抗生素的潜在靶点，并且由于基因组复制的错误调节可以促进高等生物中癌症的形成，这项工作将有助于定义抗生素靶点并完善我们对分子的理解。可能促进癌症的事件。