Visualizing the Bacterial Replisome at Single-Molecule Resolution

以单分子分辨率可视化细菌复制体

• 批准号: 8977855
• 负责人: Elizabeth Simmons Thrall
• 金额: $ 5.6万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8977855
• 关键词: Address; Architecture; Bacillus subtilis; Bacteria; Bacterial Model; Biochemical; Biochemical Genetics; Biological; Biological Assay; Cell Survival; Cells; Chimeric Proteins; Collaborations; Complex; DNA; DNA Primase; DNA biosynthesis; DNA-Directed DNA Polymerase; Disease; Enzymes; Escherichia coli; Eukaryota; Event; Genetic; Genetic study; Genomic DNA; Genomic Instability; Gram-Positive Bacteria; Image; Imaging Techniques; Immunobiology; In Vitro; Individual; Investigation; Kinetics; Laboratories; Lead; Life; Malignant Neoplasms; Measures; Mediating; Methods; Microbiology; Microscopy; Molecular; Mutation; Organism; Polymerase; Process; Proteins; Recruitment Activity; Regulation; Research; Resolution; Role; Slide; System; Tertiary Protein Structure; Ursidae Family; biochemical tools; genetic manipulation; in vivo; insight; novel; protein complex; protein protein interaction; public health relevance; reconstitution; research study; role model; single molecule; stoichiometry

DESCRIPTION (provided by applicant): The accurate and efficient replication of genomic DNA is critical for all organisms. Errors in this process can lead to mutations and ultimately to cancer. The important task of DNA replication is performed by a multi- protein complex, the replisome, which shares broadly conserved features across all domains of life. The central component of the replisome is DNA polymerase, the enzyme that synthesizes DNA with the help of processivity factors and other accessory proteins. In eukaryotes, three different polymerases are needed for chromosomal replication. This is in stark contrast to the model bacterial system, Escherichia coli, which has a single replicative polymerase. More recently, it has been discovered that two replicative polymerases, PolC and DnaE, are required in low-GC Gram-positive bacteria like Bacillus subtilis. Questions remain about the role of these polymerases and the mechanisms by which their activity is coordinated. Unlike PolC, DnaE lacks a proofreading domain and is error-prone; thus the proper regulation of DnaE activity during replication is critical for the cell. A detailed molecular-level understanding of how polymerase activity is coordinated by B. subtilis will provide insights into how replicative polymerases are regulated in more complex eukaryotic systems. This research will utilize novel single-molecule approaches to address the following specific aims: Aim 1: Determine the architecture and organizing principles of the B. subtilis replisome. It is becoming clear that the organization of the replisome in B. subtilis differs in key ways from that in E. coli, likely becaue of the need to coordinate two different polymerases. This aim will utilize in vivo single-molecule imaging of fluorescent fusion proteins to determine the copy number of important replisome components like the polymerases PolC and DnaE, the sliding clamp DnaN, and the τ subunit of the clamp loader complex. The protein-protein interactions that help to organize the replisome will be identified by making targeted mutations to domains implicated in these interactions. Aim 2: Investigate how PolC and DnaE activity is coordinated at the replication fork. In a current model for the role of PolC and DnaE, the two polymerases act sequentially on the lagging strand, meaning that polymerase exchange must occur repeatedly during replication. How such polymerase switching events are regulated and how interactions with replisome components mediate the exchange is unknown. These questions will be addressed by utilizing single-molecule imaging to measure the lifetimes of PolC and DnaE at the replication fork in live B. subtilis cells. The dynamics of individual PolC and DnaE molecules, which are obscured in ensemble biochemical experiments, will help to elucidate their roles during replication. A more targeted mechanistic investigation of polymerase exchange will be performed using an in vitro single- molecule DNA synthesis assay involving a minimally reconstituted replisome. This assay will reveal the kinetics of polymerase exchange and will help identify the protein-protein interactions involved in this process.

描述（由申请人提供）：基因组 DNA 的准确和高效复制对于所有生物体都至关重要。该过程中的错误可能导致突变并最终导致癌症。DNA 复制的重要任务是由多蛋白复合物执行的。复制体，在生命的所有领域都具有广泛保守的特征，复制体的核心成分是DNA聚合酶，这种酶在持续因子和其他辅助蛋白的帮助下合成DNA，在真核生物中，存在三种不同的聚合酶。这与具有单一复制聚合酶的模型细菌系统形成鲜明对比，最近发现低GC需要两种复制聚合酶：PolC和DnaE。与 PolC 不同，枯草芽孢杆菌等革兰氏阳性细菌的作用及其活性协调机制仍存在疑问。因此，在复制过程中正确调节 DnaE 活性对于细胞至关重要，详细了解枯草芽孢杆菌如何协调聚合酶活性将有助于了解如何在更复杂的真核系统中调节复制聚合酶。新颖的单分子方法来解决以下具体目标： 目标 1：确定枯草芽孢杆菌复制体的结构和组织原则 越来越明显的是，枯草芽孢杆菌中的复制体的组织有所不同。与大肠杆菌的关键方式不同，可能是因为需要协调两种不同的聚合酶，这一目标将利用荧光融合蛋白的体内单分子成像来确定重要复制体成分（如聚合酶 PolC 和 DnaE）的拷贝数。 、滑动钳 DnaN 和钳装载复合物的 τ 亚基将通过对涉及这些相互作用的域进行靶向突变来识别有助于组织复制体的蛋白质-蛋白质相互作用。研究 PolC 和 DnaE 活性如何在复制叉处协调。在当前 PolC 和 DnaE 作用的模型中，两种聚合酶顺序作用于滞后链，这意味着聚合酶交换必须在复制过程中重复发生。转换事件受到调节，以及与复制体成分的相互作用如何介导交换尚不清楚，这些问题将通过利用单分子成像测量活 B 中复制叉处的 PolC 和 DnaE 的寿命来解决。单个 PolC 和 DnaE 分子的动态在整体生化实验中被掩盖，将有助于阐明它们在复制过程中的作用，将使用体外单分子 DNA 合成测定进行更有针对性的聚合酶交换机制研究。最低限度重建的复制体。该测定将揭示涉及交换的聚合酶动力学，并有助于识别该过程中涉及的蛋白质-蛋白质相互作用。