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，需要许多组分的协调作用。复制缺陷可能会导致生物体的灾难性后果，因此深入了解DNA复制的机制和组成部分可以直接应用于解决与人类健康相关的问题。由于DNA复制的机制在所有生物实体中是保守的，噬菌体T7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聚合酶不能从头启动DNA链。这类重要的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，用于启动冈崎片段的合成。DNA引物酶也参与DNA解旋酶的装载、复制的调节以及引物向DNA聚合酶的传递。 这个建议的目的是调查三个主要方面的T7 DNA引发酶的功能，目前模糊采用生物物理和生物化学方法。具体而言，本建议的目的是研究的分子基础，通过调查的引物合成和移交的噬菌体T7 DNA引物酶的调节：引物合成（目标1），作用保守的氨基酸在稳定的引物模板双链体（目标2），引物移交DNA聚合酶（目标3）的引物亚结构域相互作用的作用。 从这些研究中获得的结果将提供深入了解DNA引发酶的基本功能，并应广泛适用于其他复制系统。 公共卫生相关性：DNA的复制是所有生物的基本过程。DNA引发酶是对复制起始和滞后链合成至关重要的酶;此外，它们在复制叉的协调中起着中心调节作用。我们将利用噬菌体T7 DNA复制系统的相对简单性来研究这些酶的引物合成和传递机制。

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. PUBLIC HEALTH RELEVANCE: The replication of DNA is an essential process of all living things. DNA primases are enzymes that are critical for the initiation of replication and lagging- strand synthesis; in addition they play central regulatory roles in the coordination of the replication fork. We will exploit the relative simplicity of the bacteriophage T7 DNA replication system to investigate the mechanisms of primer synthesis and handoff by these enzymes.