Replication of the lagging strand by DNA Polymerase III Holoenzyme

9303921 O‘Donnell复制大肠杆菌染色体的聚合酶，DNA聚合酶III全酶(PolIII)，由10个不同的亚基组成。作为一种全酶，polIII水解三磷酸腺苷与DNA紧密结合，使DNA合成高度进行性。全酶可分解为亚基：3亚基核心聚合酶亚基(聚合酶)、亚基(3‘-5’核酸外切酶)和0亚基、5亚基y复合体(y@xy亚基)、B亚基和t亚基。Y复合体的功能是偶联三磷酸腺苷，将B亚基传递给DNA。B亚基是一个二聚体，形状像一个环(X射线分析)，完全包围DNA。然后，B环与核心聚合酶结合，将其与DNA捆绑在一起，进行高度进行性的合成。T亚单位二聚体将两个核心聚合酶结合在一起，推测是为了同时合成双链染色体的前导和滞后链。滞后链的合成是不连续的，由大约300个片段(冈崎片段)组成。因此，每次滞后链上的polIII完成冈崎片段时，它必须能够从片段末端快速循环自身，以启动新片段的合成。然而，polIII与DNA的结合如此紧密地受到B亚单位环的影响，以至于它仍然与完整的产品DNA紧密结合，而不是循环到新的启动模板。我们最近发现了一种机制，通过这种机制，polIII可以快速循环到新的DNA模板。该机制需要一种新的polIII结构的解聚和重组，其中polIII在完成DNA模板的复制后专门脱离其B环，然后与新DNA分子上的B环重新组装。这项提议旨在发现这种聚合酶转移事件背后的详细分子基础。新的试剂和技术对拟议的工作很重要，包括对polIII的10个亚基中的每个亚基进行大量纯制剂，以及从这些单独的蛋白质重建整个全酶的能力。因此，我们建议进行一系列亚基遗漏研究，以确定哪些亚基负责将B环从polIII中分离出来，以及哪些亚基是将聚合酶转移到另一个DNA分子上的下一个B环所必需的。我们还将研究当聚合酶循环到多个模板时，留在DNA上的B环如何本身从DNA中移除，以便最终重新利用。然后，我们将开发一个复制分叉系统来研究在正在进行的双链DNA合成过程中，需要polIII的哪些亚基来与解旋酶和启动酶有效地相互作用。遗传物质是由两条相互缠绕的脱氧核糖核酸或DNA组成的。这两条DNA链包含指示细胞如何生存所需的信息(例如。Eat和Divide)，因此必须在细胞分裂之前复制它们，以便每个新细胞接收这些指令的副本。这种DNA复制的过程被称为“复制”，它是通过一系列复杂的步骤进行的，其中许多步骤还没有得到准确的定义。每一步都由不同的蛋白质分子执行，因此复制DNA需要几个“复制蛋白质”。这几种复制蛋白聚集在一起形成一个多蛋白复合体，其中每个蛋白质都占据着一个不同的位置，在整个过程中它可以执行其单独的功能。多蛋白质复合体类似于一个机器，每个齿轮执行一个功能，不同的是，这里的齿轮是蛋白质，每个蛋白质执行一个功能。最近，这个实验室发现，这种细菌的“复制机器”--大肠杆菌--的其中一个齿轮是一种形状像垫圈的蛋白质(即。环)并完全包围DNA链，以便将复制机器系在DNA上，从而使其能够有效地执行其功能。这种细菌的每一种复制蛋白质现在都可以在单独的试管中获得，我们已经学会了如何从这些单独的齿轮组装机器。有了这些单独的蛋白质试剂以及它们如何组装成一台机器的知识，这项提议的目的是确定这台复制机器在复制两条DNA时其他单独的蛋白质或齿轮的功能是什么。***

9303921 O'Donnell The polymerase that replicates the chromosome of E. coli, DNA polymerase III holoenzyme (polIII), consists of 10 different subunits. As a holoenzyme, polIII hydrolyzes ATP to bind tightly to DNA enabling highly processive DNA synthesis. The holoenzyme can be dissociated into subassemblies: the 3-subunit core polymerase @ subunit (polymerase), @subunit (3'-5' exonuclease) and 0 subunit , the 5-subunit y complex (y@@xy subunits); the B subunit and the t subunit. The function of the y complex is to couple ATP to deliver the B subunit to DNA. The B subunit is a dimer shaped like a ring (X-ray analysis) and completely encircles DNA. The B ring then binds the core polymerase acting to tether it to DNA for highly processive synthesis. The t subunit dimer binds two core polymerases together, presumably for simultaneous synthesis of both leading and lagging strands of the duplex chromosome. Synthesis of the lagging strand is discontinuous, being composed of approximately 300 fragments (Okazaki fragments). Hence, each time polIII on the lagging strand completes an Okazaki fragment it must be capable of rapidly recycling itself from the end of the fragment to initiate synthesis of new fragment. However, the polIII is bound to DNA so tightly by the B subunit ring that it remains tightly bound to a completed product DNA rather than cycling to new primed templates. We have recently discovered a mechanism whereby polIII rapidly cycles to new DNA templates. The mechanism entails a novel disaggregation of the polIII structure followed by reassembly in which the polIII disengages its B ring specifically upon completing the replication of a DNA template and then it reassembles with a B ring on a new DNA molecule. This proposal aims to discover the detailed molecular basis behind this polymerase transfer event. New reagents and technology important to the proposed work include large amounts of pure preparations of each of the 10 subunits of polIII an d the ability to reconstitute the entire holoenzyme from these individual proteins. Hence we propose to perform a series of subunit omission studies to determine which subunits are responsible for disengaging the B ring from polIII and which are needed to transfer the polymerase to the next B ring on another DNA molecule. We will also investigate how the B rings that are left on DNA as the polymerase cycles to multiple templates, are themselves removed from the DNA for eventual reutilization. Then we will develop a replication fork system to investigate which subunits of polIII are needed for efficient interaction with the helicase and primase during ongoing synthesis of both strands of duplex DNA. %%% The genetic material is in the form of two long interwound strands of deoxyribonucleic acid, or DNA. These two DNA strands contain the information needed to instruct the cell how to live (eg. eat and divide) and therefore they must be duplicated prior to cell division such that each new cell receives a copy of these instructions. This process of DNA duplication is called "replication" and it is performed in a series of complicated steps many of which have yet to be precisely defined. Each step is performed by a different protein molecule and therefore several "replication proteins" are needed to duplicate DNA. These several replication proteins assemble together to form a multiprotein complex in which each protein occupies a distinct position where it can carry out its individual function in the overall process. The multiprotein complex is analogous to a machine in which each gear performs a function, except here the gears are proteins and each protein performs a function. Recently this laboratory showed that one of the gears of this "replication machine" of the bacterium, Escherichia coli, is a protein shaped like a washer (ie. a ring) and completely encircles the strands of DNA for the purpose of tethering the replication machine down to DNA so it can eff iciently perform its function. Each of the replication proteins for this bacterium are now available in separate test tubes and we have learned how to assemble the machine from these separate gears. With these individual protein reagents and the knowledge of how they assemble into a machine, this proposal aims to determine what the function is of other individual proteins, or gears, of this replication machine as it duplicates the two strands of DNA. ***