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一个非常保守的基本过程，复制组分是潜在的抗生素靶标，复制的失调可促进多细胞生物体中的肿瘤发生。本申请检查细菌复制起始，其作为简化模型，由于存在具有单个复制起点oriC和单个复制起始蛋白DnaA的单个染色体。DnaA与oriC结合并指导复制机器的组装。通过DnaA的核苷酸结合调节DnaA活性，但由于DnaA的重要性、DnaA的自动调节以及基于oriC的质粒复制模型不能忠实地复制染色体复制，因此解剖这种调节已被证明是困难的。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）绘制B之间的相互作用。（3）研究了YabA对DnaA的调控作用和钳夹作用。为了实现这些目标，我将使用定点诱变来产生锁定为核苷酸空、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/水解在调节B中的作用。枯草杆菌DnaA-oriC相互作用和开放复合物的形成。DnaA与染色体复制起点的相互作用将使用染色质免疫沉淀和体内化学足迹法直接在细菌染色体上进行探测。用B.能够从oriC或异源的DnaA非依赖性复制起点oriN，I复制的枯草杆菌菌株将能够表征DnaA中的其它致死突变对oriC体内结合的影响，从而定义核苷酸结合在支持体内复制起始中的作用。最后，我将剖析在B中存在的类似RDA的机制。通过表征YabA和B在调节中所起的作用，对枯草芽孢杆菌进行了研究。枯草杆菌DnaA的核苷酸结合/水解，DnaA-oriC相互作用和DnaA介导的复制起始。公共卫生相关性：该应用程序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，并且由于基因组复制的错误调节可以促进高等生物的癌症形成，这项工作将有助于定义抗生素靶点并完善我们对可能促进癌症的分子事件的理解。