Primer Synthesis and Handoff by Bacteriophage T7 DNA Primase

噬菌体 T7 DNA 引物酶的引物合成和交接

• 批准号: 8464568
• 负责人: Alfredo Jose Hernandez
• 金额: $ 4.92万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8464568
• 关键词: 2-Aminopurine; Affinity; Amino Acids; Bacteriophage T7; Bacteriophages; Binding; Biochemical; Biological; Biological Models; Complex; DNA; DNA Primase; DNA Primers; DNA biosynthesis; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Defect; Enzymes; Escherichia coli; Fluorescence; Fluorescence Resonance Energy Transfer; Genes; Goals; Health; Human; Investigation; Knowledge; Label; Lead; Life; Mass Spectrum Analysis; Mediating; Methods; Molecular; Molecular Conformation; N-terminal; Nucleotides; Okazaki fragments; Organism; Play; Polymerase; Problem Solving; Process; Proteins; RNA primers; Regulation; Relative (related person); Replication Initiation; Research; Research Training; Ribonucleotides; Role; Sequence Alignment; Site; Structure; System; Testing; Zinc; base; direct application; helicase; insight

DESCRIPTION (provided by applicant): The replication of DNA is an intricate process that requires the coordinated action of many components. Defects in replication can lead to catastrophic consequences for an organism, so the acquisition of in-depth knowledge of the mechanism and components of DNA replication has direct application to solving problems related to human health. Since the mechanisms of DNA replication are conserved among all biological entities, the relatively simplicity of the replication machinery of bacteriophage T7 makes it an ideal model system to study mechanistic aspects of DNA replication. DNA primase plays a crucial role in DNA replication because DNA polymerases cannot initiate DNA chains de novo. This essential class of enzymes catalyzes the synthesis of ribonucleotides that serve as primers for DNA polymerase for the initiation of Okazaki fragment synthesis. DNA primase is also involved in loading of DNA helicase, in the regulation of replication, and handoff of the primer to DNA polymerase. The objective of this proposal is to investigate three major aspects of T7 DNA primase function that are currently obscure employing biophysical and biochemical methods. Specifically, the aims of this proposal are to study the molecular bases for the regulation of primer synthesis and handoff by bacteriophage T7 DNA primase through the investigation of: the role of primase subdomain interactions in primer synthesis (Aim 1), the role conserved amino acid in the stabilization of the primer-template duplex (Aim 2), and the process of primer handoff to DNA polymerase (Aim 3). The results obtained from these studies will provide insight into fundamental functions of DNA primase and should be broadly applicable to other replication systems.

描述(申请人提供)：DNA的复制是一个复杂的过程，需要许多组件的协调行动。复制中的缺陷会给生物体带来灾难性的后果，因此深入了解DNA复制的机制和组成部分对于解决与人类健康有关的问题具有直接的应用价值。由于DNA复制机制在所有生物实体中都是保守的，T7噬菌体的复制机制相对简单，使其成为研究DNA复制机制的理想模型系统。DNA聚合酶在DNA复制中起着至关重要的作用，因为DNA聚合酶不能重新启动DNA链。这类重要的酶催化合成核糖核苷酸，作为启动冈崎片段合成的DNA聚合酶的引子。DNA底物酶还参与DNA解旋酶的装载，复制的调节，以及将底物传递给DNA聚合酶。该提案的目的是用生物物理和生化方法研究目前尚不清楚的T7DNA启动子酶功能的三个主要方面。具体地说，本研究的目的是通过以下几个方面的研究来研究T7噬菌体DNA底物酶调节底物合成和转移的分子基础：底物亚区相互作用在底物合成中的作用(目的1)，保守氨基酸在底物-模板双链稳定中的作用(目的2)，以及底物移交给DNA聚合酶的过程(目的3)。这些研究的结果将为深入了解DNA引物酶的基本功能提供帮助，并应广泛适用于其他复制系统。