DNA-PROTEIN INTERACTIONS IN DNA-REPLICATION CONTROL

T4噬菌体DNA聚合酶（基因43的产物），T4 螺旋不稳定蛋白（基因32蛋白），和其他五个T4- 诱导蛋白质（基因41、44、45、61和62的产物）是 蛋白质复合体的基本组成部分， 叉子 44/62和45蛋白是DNA聚合酶的一部分 “全酶”和称为“聚合酶辅助蛋白”。 基因 41蛋白是一种DNA解旋酶，它利用GTP水解能量移动 快速沿着DNA;它形成一个移动的复合物，称为 “primosome”与基因61蛋白，这是引发酶， 合成五核糖核苷酸引物， 复制叉落后链上的片段。 在体外 反应需要所有七种纯化的蛋白质（加上脱氧核糖， 和核糖核苷三磷酸），复制叉通过 纯化的双链DNA模板具有接近体内速率， 两个人 我们的实验表明， 分叉处落后链上的DNA聚合酶分子。 这 循环被认为涉及DNA之间的直接连接， 聚合酶全酶复合物存在于前导和滞后 链，以及与DNA解旋酶的密切相互作用。 的 整个复合体作为一个单元工作;例如，RNA引物合成 似乎被延迟，直到滞后链DNA聚合酶分子 在复杂的完成每个冈崎片段，并从释放 DNA. 这项建议的第一个主要目标是制定一个更详细的 理解刚才描述的一致的叉运动反应。 我们认为，辅助蛋白形成一个滑动钳， 释放机制，从而使聚合酶全酶从一个 非解离形式到最小后快速解离形式 停止时间已超过。 将寻找突变辅助蛋白， 使聚合酶全酶始终保持结合。 DNA复合物 由这种全酶形成的DNA将用于DNA足迹， 结晶研究，长期目标是确定 复制蛋白复合物的完整三维结构。 该提案的第二个主要目的是识别、净化和 表征重建该过程所需的所有T4蛋白 的跨损伤DNA合成。 暂时的，重组- 在第二个DNA模板上介导转换为保守DNA合成 据信发生在可能需要（在 除复制蛋白外，T4 uvsX（recA样）、uvsY、dda和 基因59蛋白质，迄今为止，在这个实验室的特点，加上在 至少有一个组件丢失。

The T4 bacteriophage DNA polymerase (the product of gene 43), the T4 helixdestabilizing protein (the gene 32-protein), and five other T4- induced proteins (the products of genes 41, 44, 45, 61, and 62) are the fundamental components of the protein complex that moves a replication fork. The 44/62 and 45 proteins are part of the DNA polymerase "holoenzyme" and are termed "polymerase accessory proteins". The gene 41 protein is a DNA helicase that uses GTP hydrolysis energy to move rapidly along the DNA; it forms a moving complex known as the "primosome" with the gene 61 protein, which is the primase that synthesizes the pentaribonucleotide primers that start each Okazaki fragment on the lagging strand of a replication fork. In an in vitro reaction requiring all seven of these purified proteins (plus deoxyribo- and ribonucleoside triphosphates), replication forks move through purified double-stranded DNA templates with near in vivo rates and fidelities. Our experiments have revealed that there is a continuous recycling of the DNA polymerase molecule on the lagging strand at the fork. This recycling is believed to involve a direct connection between the DNA polymerase holoenzyme complexes present on the leading and lagging strands, as well as a close interaction with the DNA helicase. The entire complex works as a unit; for example, RNA primer synthesis appears to be delayed until the lagging strand DNA polymerase molecule in the complex finishes each Okazaki fragment and is released from the DNA. The first major aim in this proposal is to develop a more detailed understanding of the concerted fork movement reaction just described. We believe that the accessory proteins form a sliding clamp with a timed release mechanism, so that the polymerase holoenzyme converts from a non-dissociating form to a rapidly dissociating form after a minimum stall time is exceeded. Mutant accessory proteins will be sought that keep the polymerase holoenzyme bound at all times. The DNA complexes formed by this holoenzyme will be used for DNA footprinting and crystallization studies, with the long-range aim of determining the complete three-dimensional structure of the replication protein complex. The second major aim of this proposal is to identify, purify, and characterize all of the T4 proteins required to reconstruct the process of translesion DNA synthesis in vitro. A temporary, recombination- mediated switch to conservative DNA synthesis on a second DNA template is believed to occur, in a reaction that is likely to require (in addition to replication proteins) the T4 uvsX (recA-like), uvsY, dda and gene 59 proteins thus far characterized in this laboratory, plus at least one missing component.