Subunit Arrangement within a Replicative Complex

9513248麦克亨利DNA聚合酶III全酶是大肠杆菌的复制复合物。大肠杆菌，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。 它们都由一个特殊的复制聚合酶，结构相同的滑动夹与聚合酶和夹具设置装置。 在大肠这些组件对应于DNA聚合酶III（））、（“滑动夹”和5-蛋白DnaX夹设置装置。 全酶含有10种不同的亚基，所有亚基都可以从高产菌株中获得100 mg的量。 这种复制复合物表现出许多区别于简单聚合酶的特性。 这些特性包括高延伸速率、在DNA模板上形成ATP依赖性高度加工性钳的能力、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起作用的能力。 使用放置在长引物内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）和持续合成因子（核苷酸22）。 在该提案中，概述了扩展该分析的实验，以识别亚基与模板的接触，特别是在引物前面和模板内具有ca短引物的区域。10个核苷酸，如用于引发DNA复制的那些。 将光反应性核苷酸放置在设计序列的模板中的特定位置将允许复制复合物通过添加有限数量的dNTP而“走”到特定位置。 这将允许在复制复合物内的组成和接触的变化被确定为从起始到延伸的过程。 亚基相对于复制复合物中其它亚基的定位将通过化学蛋白质-蛋白质交联来确定。 最初的重点将放在“零长度”交联剂EDC上。 将在序列水平上分析这些阿托伐他汀诱导的亚基间交联，以使亚基-亚基接触区域局部化。 荧光能量转移将用于确定亚基之间的距离。 主要关注的是不相互交联的亚基之间的距离。 同时测定这些亚基到引物末端和α亚基的距离将允许三角测量和将所研究的组分放置在复制复合物内。 这些实验得到了我们证明的从引物中的3 '-倒数第二个核苷酸定位亚基65 A的能力的支持，该位置通过光交联验证。 此外，试点实验将进行合作使用电子显微镜检查的复制复合物的结构。 最初的重点将放在二聚体聚合酶假说的直接视觉测试上。 这项拟议的工作将产生第一个详细的结构，一个原型的复制复合体在亚基组织的水平。 %%% 在细胞分裂之前复制染色体的10蛋白质装置在细菌和高等细胞之间是非常保守的。这项研究的目的是确定一个原型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碱基的类似物放置在模型染色体中的特定点上，当用光闪烁时，所述DNA碱基的类似物化学地附着到蛋白质上，以确定复制蛋白质相对于DNA在静态和主动延伸模式下的线性排列;（ii）直接化学蛋白质-蛋白质交联以确定哪些蛋白质彼此接触，（iii）荧光能量转移，一种使用光谱（光）尺来确定彼此不接触的组件之间的距离的技术，以及（iv）电子显微镜直接可视化蛋白质和DNA的相对位置。 这些互补的方法将允许一个原型的复制复合物的几何形状的描述，并允许特定的实验设计，以测试蛋白质亚基的功能暗示其在复合物内的位置（S）。 ***

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. ***