INITIATION OF LAGGING-STRAND DNA SYNTHESIS

拟议的工作将继续我们实验室的努力，以详细说明 在滞后链DNA合成和启动过程中发生的事件 随后对DNA上的启动装置进行维护。二 PBR322 DNA的区域已被证明作为效应DNA 复制因子Y的单链DNA依赖的ATPase活性 (齐普尔斯基和玛丽斯，PNAS，77,6521-6525(1980))，一株预引发菌 指导原始体组装的复制蛋白。我们有 证实了pBR322 DNA的Y因子效应位点可以作为 克隆成单链时互补链合成的起源 噬菌体载体(Zipusky和Mariers，PNAS，78,6111-6115(1981))。这个 拟开展的工作可分为两大部分。我们将首先研究 Y因子与其效应部位在分子水平上的相互作用。 通过使用DNA足迹、甲基化增强和体外 突变构建我们将精确地确定一个因子中的那些碱基 Y效应器部位，这是起源功能所必需的。这项工作将 确定因子Y与其效应DNA相互作用的机制 并定义了DNA区域作为 复制的滞后链起始点。建议的第二部分 工作本质上是生化的。大于Rf DNA的ss(C)的重组 利用重组Y-位点fl噬菌体DNA合成纯化蛋白 因为模板将允许我们确定涉及哪些蛋白质， 原始体运动方向及ATP水解酶的作用 解开双链DNA过程中的蛋白质(Y因子和dna B蛋白) 复制。

The proposed work will continue our laboratory's effort to detail the events that occur during the initiation of lagging-strand DNA synthesis and the subsequent maintenance of the priming apparatus on the DNA. Two regions of pBR322 DNA have been shown to act as effector DNAs for the singlestrand DNA-dependent ATPase activity of replication factor Y (Zipursky and Marians, PNAS, 77, 6521-6525 (1980)), a pre-priming E. coli replication protein which directs the assembly of the primosome. We have demonstrated that the factor Y effector sites of pBR322 DNA can act as origins of complementary strand synthesis when cloned into single-strand phage vectors (Zipursky and Marians, PNAS, 78, 6111-6115(1981). The proposed work can be divided into two major sections. We will first study the interaction of factor Y with its effector sites on a molecular level. Through the use of DNA footprinting, methylation enhancement and in vitro mutant construction we will determine precisely those bases within a factor Y effector site which are required for origin function. This work will define the mechanism of interaction between factor Y and its effector DNAs and define the requirements for a region of DNA to function as a lagging-strand origin of replication. The second section of the proposed work is biochemical in nature. Reconstitution of ss(c) greater than RF DNA synthesis with purified proteins using the recombinant Y-site-fl phage DNAs as templates will allow us to determine which proteins are involved, the direction of primosome movement, and the role of the ATP hydrolyzing proteins (factor Y and the dnaB protein) in unwinding duplex DNA during replication.