INITIATION OF LAGGING-STRAND DNA SYNTHESIS

拟议的工作将继续我们的实验室的努力，详细说明 在滞后链DNA合成起始期间发生的事件， 随后对DNA上的引发装置进行维护。 两 pBR 322 DNA的区域已经显示出作为效应DNA， 复制因子Y单链DNA依赖性ATP酶活性 （Zipursky和Marians，PNAS，77，6521-6525（1980）），预引发E.杆菌 指导原体装配的复制蛋白。 我们有 证明了pBR 322 DNA的Y因子效应位点可以作为 当克隆到单链中时互补链合成的起源 噬菌体载体（Zipursky和Marians，PNAS，78，6111-6115（1981）。 的 拟议的工作可分为两大部分。 我们先来研究一下 因子Y与其效应位点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 突变体的构建我们将精确地确定这些碱基在一个因素 Y效应子位点，其是起源功能所需的。 这项工作将 明确Y因子与其效应DNA之间的相互作用机制 并定义了DNA区域发挥作用的要求， 滞后链复制起点。 第二节建议 工作本质上是生物化学的。 ss（c）的复溶大于RF DNA 使用重组Y-位点-fl噬菌体DNA合成纯化的蛋白质 因为模板将使我们能够确定哪些蛋白质参与， 启动子运动的方向，以及ATP水解的作用 蛋白质（因子Y和dnaB蛋白）在解旋双链体DNA过程中 复制的

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.