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-蛋白- 诱导蛋白(基因41、44、45、61和62的产物)是 移动复制的蛋白质复合体的基本成分 叉子。44/62和45蛋白是DNA聚合酶的一部分。 “全酶”，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水解能进行运动。 沿着DNA快速移动；它形成一个移动的复合体，称为 “原始体”与基因61蛋白有关，这是启动酶 合成了从每个冈崎开始的五核苷酸引物 复制叉子的滞后链上的碎片。在体外实验中 反应需要所有这些纯化的蛋白质(加上脱氧核糖核酸- 和核糖核苷三磷酸)，复制叉子穿过 纯化的双链DNA模板具有接近体内的速度和 忠诚度。 我们的实验表明，存在着一个连续的循环 分叉处滞后链上的DNA聚合酶分子。这 回收被认为涉及DNA之间的直接联系 聚合酶全酶复合体存在于领先和落后 链，以及与DNA解旋酶的密切相互作用。这个 整个复合体作为一个单元工作；例如，RNA引物合成 似乎被推迟到滞后的链DNA聚合酶分子 在复合体中完成每个Okazaki片段并从 DNA这项提案的第一个主要目标是制定一个更详细的 对刚才描述的协调叉子运动反应的理解。 我们认为，辅助蛋白形成了一个滑动的夹子，带有一个定时的 释放机制，使聚合酶全酶从 从非解离形式到快速解离形式 已超过停顿时间。将寻找突变的辅助蛋白 任何时候都要保持聚合酶全酶结合。DNA复合体 由这种全酶形成的将用于DNA足迹和 结晶研究，长期目标是确定 复制蛋白复合体的完整三维结构。 该提案的第二个主要目标是识别、净化和 鉴定重建过程所需的所有T4蛋白 体外跨损伤DNA合成的研究。一种暂时的，重组- 在第二个DNA模板上介导的向保守DNA合成的转换 据信发生在一种可能需要(在 除复制蛋白外)T4uvsX(类recA)、uvsY、dda和 到目前为止，基因59蛋白在这个实验室中的特征，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.