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体，负责大部分染色体的合成。所有细胞系统的复制复合体是密切相关的。它们都由一种特殊的复制聚合酶、与该聚合酶相关联的结构相同的滑动夹子和夹子设置装置组成。在大肠杆菌中，这些成分对应于DNA聚合酶III())、(“滑动夹”)和5-蛋白DNAX夹设置装置。全酶包含10个不同的亚基，所有亚基都是从高产菌株中获得的100毫克量。这种复制复合体表现出许多区别于更简单的聚合酶的性质。这些特性包括高伸长率，在DNA模板上形成依赖于ATP的高度进行性夹子的能力，以及作为不对称二聚体发挥作用的能力，具有明显的领先和滞后链聚合酶。利用放置在长引物中独特位置的光反应核苷酸，我的实验室已经在起始复合体中定位了(催化亚单位(3‘-核苷酸1-13)、((DNAX)-复合体(在第18位核苷酸处接触)和处理因子(第22位核苷酸)。在本提案中，概述了扩展这种分析的实验，以便鉴定亚基与模板的接触，特别是在引物前面和模板内具有约10个核苷酸的短引物的区域，例如用于启动DNA复制的那些。将光反应核苷酸放置在设计序列的模板中的特定位置，将允许通过添加有限数量的dNTP将复制复合体“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。亚基在复制复合体中相对于其他亚基的位置将由化学蛋白质-蛋白质交联性决定。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链将在序列水平上进行分析，以允许亚基-亚基接触的区域被定位。荧光能量转移将被用来确定亚基之间的距离。主要的关注点将是相互之间没有交联链的亚基之间的距离。同时测定这些亚基到引物末端和(亚基)的距离将允许三角测量和将研究的成分放置在复制复合体中。这些实验得到了我们证明的从3‘-倒数第二核苷酸中定位(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，还将合作进行试点实验，使用电子显微镜检查复制复合体的结构。最初的焦点将放在对二聚体聚合酶假说的直接目测上。这项拟议的工作将在亚基组织水平上产生原型复制复合体的第一个详细结构。在细胞分裂前复制染色体的10蛋白装置在细菌和高等细胞之间非常保守。这项拟议的研究的目的是确定原型DNA复制机器的物理排列和组件的接触。要做到这一点，方法是：(1)将DNA碱基类似物放置在模型染色体上的特定点上，这些碱基在光照射下以化学方式连接到蛋白质上，以确定复制蛋白质在静态和主动伸展模式下相对于DNA的线性排列；(2)直接化学蛋白质-蛋白质交联以确定哪些蛋白质相互接触，(3)荧光能量转移，这种技术使用光谱(光)尺来确定相互不接触的成分之间的距离，以及(4)电子显微镜可以直接观察蛋白质和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结构，并允许设计特定的实验来测试蛋白质亚单位在复合体中的位置(S)所暗示的功能。***

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. ***