Subunit Arrangement within a Replicative Complex

DNA聚合酶III全酶是大肠杆菌的复制复合体，负责大部分染色体的合成。所有细胞系统的复制复合体都是密切相关的。它们都由一种特殊的复制聚合酶、结构相同的滑动夹和夹紧装置组成。在大肠杆菌中，这些成分对应于DNA聚合酶III(((((),（“滑动钳”）和5蛋白DnaX钳设置装置。这种全酶含有10种不同的亚基，每100毫克都可以从过量生产的菌株中获得。这种复制复合体表现出许多不同于简单聚合酶的特性。这些特性包括高延伸率，在DNA模板上形成atp依赖的高度过程钳的能力，以及作为具有可区分的前导链和滞后链聚合酶的不对称二聚体的能力。使用放置在长引物独特位置的光反应性核苷酸，我的实验室已经定位了起始复合物内的催化亚基（3'-核苷酸1-13），(DnaX)-复合物（在核苷酸18处接触）和加工因子（核苷酸22）。在本提案中，实验概述了扩展该分析，以确定与模板的亚基接触，特别是在引物前面和模板内的区域，使用大约10个核苷酸的短引物，例如用于引物DNA复制的引物。将光反应性核苷酸放置在设计序列模板中的特定位置，将允许复制复合体通过添加有限数量的dntp“行走”到特定位置。这将允许在从起始到延伸的过程中确定复制复合体内的组成和接触的变化。在复制复合体中，亚基相对于其他亚基的定位将由化学蛋白质-蛋白质交联决定。最初的重点将放在“零长度”交联剂EDC上。这些碳二亚胺诱导的亚基间交联将在序列水平上进行分析，以允许亚基-亚基接触区域被定位。荧光能量转移将用于确定亚基之间的距离。主要的焦点将放在不相互交联的亚基之间的距离上。同时确定这些亚基到引物末端和亚基的距离将允许三角测量和在复制复合体内放置所研究的成分。这些实验得到了我们在引物中3'-前倒数核苷酸上定位（亚基65a）的能力的支持，这一位置通过光交联得到了验证。此外，试点实验将进行合作，使用电子显微镜来检查复制复合体的结构。最初的重点将放在二聚体聚合酶假说的直接视觉测试上。这项提议的工作将在亚单位组织水平上产生原型复制复合体的第一个详细结构。在细胞分裂之前复制染色体的10个蛋白质装置在细菌和高等细胞之间是密切保守的。本研究的目的是确定一个典型的DNA复制机器的组成部分的物理安排和接触。这将通过(i)将DNA碱基类似物放置在模型染色体的特定点上，该模型染色体在用光闪烁时化学地附着在蛋白质上，以确定在静态和主动延伸模式下复制蛋白质相对于DNA的线性排列；（ii）直接化学蛋白质-蛋白质交联，以确定哪些蛋白质相互接触；（iii）荧光能量转移，一种使用光谱（光）尺确定不相互接触的成分之间距离的技术；（iv）电子显微镜，直接观察蛋白质和DNA的相对位置。这些互补的方法将允许描述原型复制复合体的几何形状，并允许设计特定的实验来测试蛋白质亚基在复合体内的位置所隐含的功能。＊＊＊

9513248 McHenry The DNA polymerase III holoenzyme is the replicative complex of E. coli, responsible for the synthesis of the majority of the chromosome. The replicative complexes of all cellular systems are closely related. They all consist of a special replicative polymerase, structurally identical sliding clamps with which the polymerase associates and a clamp setting apparatus. In E. coli, these components correspond to the DNA polymerase III ((((), the ( "sliding clamp", and the 5-protein DnaX clamp setting apparatus. The holoenzyme contains 10 different subunits, all available in 100 mg quantities from overproducing strains. This replicative complex exhibits many properties that distinguish it from simpler polymerases. These properties include a high rate of elongation, the ability to form an ATP-dependent highly processive clamp on the DNA template, and the ability to function as an asymmetric dimer with distinguishable leading and lagging strand polymerases. Using photoreactive nucleotides placed at unique positions within long primers, my laboratory has located the ( catalytic subunit (3'-nucleotides 1-13), the ( (DnaX)-complex (contact at nucleotide 18) and the processivity factor (nucleotide 22) within the initiation complex. In this proposal, experiments are outlined to extend this analysis in order to identify subunit contacts with the template, particularly in front of the primer and regions within the template with short primers of ca. 10 nucleotides such as those used to prime DNA replication. Placement of photoreactive nucleotides at specific locations in templates of designed sequence will permit the replicative complex to be "walked" into specific positions by the addition of a limited number of dNTPs. This will permit changes in the composition and the contacts within the replicative complex to be determined as it progresses from initiation to elongation. The positioning of subunits relative to others within the replicative complex will be determined by chem ical protein-protein cross-linking. Initial focus will be on the "zero-length" cross-linker EDC. These carbodiimide-induced intersubunit cross-links will be analyzed at the sequence level to permit regions of subunit-subunit contact to be localized. Fluorescence energy transfer will be used to determine the distance between subunits. Primary focus will be on the distances between subunits that do not cross-link to one another. Concurrent determination of the distances of these subunits to the primer terminus and the ( subunit will permit triangulation and placement of the studied components within the replicative complex. These experiments are supported by our demonstrated ability to locate the ( subunit 65 A from the 3'-antepenultimate nucleotide in primers, a position verified by photocross-linking. Additionally, pilot experiments will be conducted collaboratively using electron microscopy to examine the structure of the replicative complex. Initial focus will be upon a direct visual test of the dimeric polymerase hypothesis. This proposed work will yield the first detailed structure of a prototypical replicative complex at the level of subunit organization. %%% The 10-protein apparatus that duplicates chromosomes prior to cell division is closely conserved between bacterial and higher cells. The purpose of this proposed study is to determine the physical arrangement and the contacts of the components of a prototypical DNA replicative machine. This will be accomplished by (i) placing analogs of DNA bases at specific points in a model chromosome that chemically attach to proteins when flashed with light to determine the linear arrangement of the replication proteins relative to DNA both in a static and an active elongation mode; (ii) direct chemical protein-protein cross-linking to determine which proteins touch one another, (iii) fluorescence energy transfer, a technique that uses a spectroscopic (light) ruler to determine the distances between the components that do not contact one another and (iv) electron microscopy to directly visualize relative positions of proteins and DNA. These complementary methods will permit a description of the geometry of a prototypical replicative complex and permit the design of specific experiments to test the function of protein subunits implied by their position(s) within the complex. ***