Primer Synthesis and Handoff by Bacteriophage T7 DNA Primase

描述（由申请人提供）：DNA的复制是一个复杂的过程，需要许多成分的协调作用。复制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复制的机制在所有生物实体中都是保守的，因此噬菌体T7的复制机制相对简单，使其成为研究DNA复制机制方面的理想模型系统。DNA引物酶在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。这类酶催化核糖核苷酸的合成，核糖核苷酸作为DNA聚合酶的引物，用于启动冈崎片段合成。DNA引物酶还参与DNA解旋酶的装载、复制的调控以及引物与DNA聚合酶的交接。本提案的目的是研究T7 DNA引物酶功能的三个主要方面，目前尚不清楚采用生物物理和生化方法。具体来说，本课题的目的是通过研究引物酶亚结构域相互作用在引物合成中的作用（Aim 1）、保守氨基酸在引物-模板双链稳定中的作用（Aim 2）以及引物与DNA聚合酶的交接过程（Aim 3）来研究噬菌体T7 DNA引物酶调控引物合成和交接的分子基础。从这些研究中获得的结果将为DNA引物酶的基本功能提供见解，并应广泛适用于其他复制系统。

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.