INITIATION OF LAGGING-STRAND SYNTHESIS

复制叉子是一个复杂的结构。20多种不同的 原核酸体很可能在一台酶机器中共同运行，该机器具有 被称为复制体。在大肠杆菌中，这种蛋白质 由原始体蛋白质和DNA组成的集合体 聚合酶III全酶(本身由10个亚基组成)在 亲本DNA以1000nT/秒的速度，同时解开模板和 以协调的方式合成新生的领先和落后链 时尚。我们的目标是了解这些复制蛋白是如何 聚集在DNA上，以照亮持有 复制分叉在一起，并确定每个蛋白质如何在 复制分叉有助于半保守的有序过程 DNA复制。为此，我们研究：i)独立的活动 单个复制蛋白，II)各种部分的性质 复制完全互补的子集催化的反应 分叉蛋白，III)影响的控制电路和参数 重组复制叉催化Okazaki片段合成 纯化的原始体蛋白和DNA聚合酶III的体外研究 以及iv)缺乏的大肠杆菌突变株的表型。 在PriA、Prib和PrIC复制蛋白中。 我们对复制分叉作用的研究使我们能够开发一个模型 描述了冈崎片段合成的控制。这是它的一个关键特征 模型是DNAB(复制叉解旋酶)和 DNAG(启动酶)，以及DNAB和DNAC(一种原始体蛋白)之间的连接。 模型的这些方面将通过分离新的突变体进行测试 在相互作用中有缺陷的蛋白质。这些将是 与现有的突变蛋白一起在重组的 复制分叉系统对冈崎片段合成的影响。 DNA聚合酶III各亚组分作用的研究 全酶提示亚基在以下方面有不同的参与： 一)新生的链合成，二)先导-和 滞后链聚合酶复合体，III)预引发和起始 在滞后链上的新引物末端形成复合体，以及 IV)在介导滞后链聚合酶在 冈崎片断合成的终止及其向新的过渡 启动子末端。Pol III HE亚基在分叉点的作用将 通过重组具有单独纯化的复制分叉来定义 亚单位。 蛋白质和DNA链之间需要精确的协调 复制分叉，以及特定蛋白质-蛋白质的重要作用 复制分叉作用中的相互作用，暗示着一个定义的分子 建筑。我们将研究存在于 使用代谢性标记的复制蛋白的复制叉子 高比活性，凝胶位移分析，电子显微镜和 免疫电子显微镜。 我们关于复制分叉操作的模型的有效性将在 通过研究DNAG在体内的Okazaki片段合成 生产是可以控制的。PRIA、PRIB和PRIC的作用 原始体蛋白在细胞复制中的作用及其原因 灭活引发SOS反应，将通过研究冈崎来探讨 PriA、Prib和PrIC中的片段合成和复制分叉运动 菌株。

The replication fork is a complex structure. Upward of 20 different protomers are likely to operate together in an enzyme machine that has been termed the replisome. In Escherichia coli, this protein conglomerate, which is made up of primosomal proteins and the DNA polymerase III holoenzyme (itself composed of 10 subunits), moves on the parental DNA at 1000 nt/sec, simultaneously unwinding the template and synthesizing the nascent leading- and lagging-strands in a coordinated fashion. It is our goal to understand how these replication proteins assemble on the DNA, to illuminate the interactions that hold the replication fork together, and to determine how each protein at the replication fork contributes to the orderly process of semi-conservative DNA replication. To do so, we study: i) The independent activities of individual replication proteins, ii) the nature of various partial reactions catalyzed by subsets of the complete complement of replication fork proteins, iii) the control circuits and parameters that affect Okazaki fragment synthesis catalyzed by replication forks reconstituted in vitro with purified primosomal proteins and the DNA polymerase III holoenzyme, and iv) the phenotypes of E. coli mutant strains deficient in the PriA, PriB, and PriC replication proteins. Our studies on replication fork action have allowed us to develop a model describing control of Okazaki fragment synthesis. A key feature of this model is the interaction between DnaB (the replication fork helicase) and DnaG (the primase), and between DnaB and DnaC (a primosomal protein). These aspects of the model will be tested by isolating new mutant proteins that are defective in their interactions. These will be examined, along with existing mutant proteins, in the reconstituted replication fork system for their affect on Okazaki fragment synthesis. Studies on the action of the subassemblies of the DNA polymerase III holoenzyme have suggested differential participation of the subunits in: i) nascent strand synthesis, ii) the interaction between the leading- and lagging-strand polymerase complex, iii) preinitiation and initiation complex formation on the new primer terminus on the lagging strand, and iv) in mediating the disassembly of the lagging-strand polymerase after termination of Okazaki fragment synthesis and in its transit to the new primer terminus. The roles of the Pol III HE subunits at the fork will be defined by reconstituting replication forks with individually purified subunits. The precise coordination required between proteins and DNA strands at the replication fork, and the important role of specific protein-protein interactions in replication fork action, implies a defined molecular architecture. We will study the protein-DNA structures present at the replication fork using replication proteins labelled metabolically to high specific activity, gel shift analysis, electron microscopy and immunoelectron microscopy. The validity of our models on replication fork action will be tested in vivo by studying Okazaki fragment synthesis in strains where DnaG production can be controlled. The role of the PriA, PriB, and PriC primosomal proteins in cellular replication, and the reason that PriA inactivation induces the SOS response, will be probed by studying Okazaki fragment synthesis and replication fork movement in priA, priB, and priC strains.