DNA-PROTEIN INTERACTIONS IN DNA-REPLICATION CONTROL

我们正试图使用单独纯化的蛋白质成分， T4噬菌体DNA复制装置， 在完全定义的体外系统中的复制过程。 而 复制叉运动过程已经忠实地重建， 这一过程的许多机械细节都很清楚， 在T4复制起点启动复制叉的进程没有 却被成功复制。 急诊大肠杆菌宿主RNA聚合酶发挥着 在后一过程中的作用，沿着T4 ATP依赖的II型DNA 拓扑异构酶和其他可疑但未知的噬菌体蛋白。 使用几种不同的检测方法，我们将尝试定义缺失的 组件，以获取源相关复制分叉 体外启动。 我们还将使用一种新的克隆策略来定义 构成主要T4复制起点的确切DAN序列。 还将进行几项旨在了解 T4多酶复合物的详细结构， 复制叉，包括它与DNA的相互作用和细节， 使滞后链DNA聚合酶分子重新聚合的过程， 连续几轮的冈崎片段合成。 最后利用 纯化的T4 uvsX和uvsY蛋白，我们将尝试重组 从基因重组开始复制叉的过程 在T4感染后期的中间体。 所有这些研究都 旨在增加我们对基本遗传学的基本知识， 所有细胞共有的机制，它们对于理解 在正常和疾病状态下的细胞行为。

We are attempting to use the individually purified protein components of the T4 bacteriophage DNA replication apparatus to reconstitute the entire replication process in a completely defined in vitro system. While the replication fork movement process have been faithfully reconstituted, and many of the mechanistic details of this process are well understood, the process that initiates replication forks at T4 replication origins has not yet been successfully reproduced. The E. coli host RNA polymerase plays a role in the latter process, along with the T4 ATP dependnet type II DNA topoisomerase and other suspected but unknown bacteriophage proteins. Using several different assays, we will attempt to define the missing components, as required to obtain origin-dependent replication fork initiation in vitro. We will also use a novel cloning strategy to define the exact DAN sequences that constitute a primary T4 replication origin. Several experiments will also be pursued that are aimed at understanding the detailed structure of the T4 multienzyme complex that moves a replication fork, including its interactions with DNA and the details of the process that recycles the lagging strand DNA polymerase molecule for successive rounds of Okazaki fragment synthesis. Finally, using the purified T4 uvsX and uvsY proteins, we will attempt to reconstitute the process that initiates replication forks from genetic recombination intermediates at late times of T4 infection. All of these studies are aimed at increasing our fundamental knowledge of the basic genetic mechanisms common to all cells, and they are important to an understanding of cell behavior in both normal and diseased states.