DNA Helicases in DNA Replication and Repair

本研究的目的是了解DNA复制起点转化为复制叉的机制，以及叉处的蛋白质如何组织以协调前导链和滞后链的合成。 其基本假设是，在复制叉处有一个蛋白质的超组装体，它的功能就像一台机器，其运动部件是蛋白质。 不太确定的是，是否有一个单一的、多蛋白质的组装体在起源处以协调一致的方式改变构象，以影响分叉进程的每个阶段，或者是否有许多较小的机器，每个机器都被分配了不同的特定任务，并且只是暂时与分叉相关联。 要解决这样一个复杂的问题，就需要把它分解成部分反应。 在即将到来的授权期内，将对DNA 2解旋酶及其在起始和延伸中的作用进行深入研究。这些基本的酵母生物学实验将与强大的非洲爪蟾卵提取物的实验相结合，在严格的细胞周期控制下，在体外重演启动和延长。非洲爪蟾的体外复制系统忠实地模仿体内的事件，并已非常有用的工作，在精子染色质上的起始蛋白的组装顺序。 该系统的一个重要特征是卵提取储备复制蛋白，并且能够在体外非常快速和广泛地复制。 因此，它们也提供了研究真核染色体复制叉的唯一现有的体外复制系统。 在此之前，在该实验室中鉴定了酵母中DNA复制所必需的第一个DNA解旋酶。 为了利用酵母分子生物学和遗传学以及非洲爪蟾体外复制系统，克隆了酵母解旋酶的非洲爪蟾同源物，称为DNA 2。 现在将在存在和不存在Xdna2p的情况下分析非洲爪蟾提取物中DNA复制的每个阶段。 将遵循在存在和不存在Xdna2p的情况下在细胞周期期间复制装置的组装和拆卸。 DNA 2缺陷的酵母突变体对X射线和MMS敏感。 由于非洲爪蟾提取物已被证明是有用的X射线诱导的损伤的修复的研究，非洲爪蟾提取物将被用于研究Xdna 2的分子贡献。研究细胞周期和复制叉的调控形成和激活对于理解细胞增殖的调控具有重要意义。 DNA复制周期也是发育和分化过程中调控的目标。 这项研究有望为这些过程提供重要的见解。

J. CampbellThe goal of this research is to understand the mechanism by which an origin of DNA replication is converted into a replication fork and how the proteins at the fork are organized to coordinate the synthesis of the leading and lagging strands. The underlying assumption, which, while plausible, remains to be proved, is that there is a superassembly of proteins at the replication fork and that it functions like a machine whose moving parts are proteins. What is less certain is whether there is a single, multiprotein assembly at origins that changes conformation in a concerted manner to effect each stage of fork progression or if there are a number of smaller machines each assigned a different specific task and only transiently associated with the fork. To attack such a complex problem requires dividing it into partial reactions. In the coming granting period, the DNA2 helicase and its role in initiation and elongation will be studied in depth. These fundamental biological experiments in yeast will be combined with experiments in the powerful Xenopus laevis egg extracts, that recapitulate both initiation and elongation in vitro under strict cell cycle control. The Xenopus in vitro replication system faithfully mimics in vivo events and has been very useful in working out the order of assembly of initiation proteins onto the sperm chromatin. An important feature of this system is that the egg extracts stockpile replication proteins and are capable of very rapid and extensive replication in vitro. Thus, they also provide the only existing in vitro replication system for studying eukaryotic chromosomal replication forks. Previously, the first DNA helicase essential for DNA replication in yeast was identified in this laboratory. In order to take advantage of both yeast molecular biology and genetics and Xenopus in vitro replication system, the Xenopus homolog of the yeast helicase, called Dna2, was cloned. Each stage of DNA replication in Xenopus extracts will now be analyzed in the presence and absence of Xdna2p. The assembly and disassembly of the replication apparatus during the cell cycle in the presence and absence of Xdna2p will be followed. Yeast mutants defective in DNA2 are sensitive to X-rays and MMS. Since the Xenopus extracts have been shown to be useful for studies of repair of X-ray induced damage, the Xenopus extracts will be used to study the molecular contribution of Xdna2. Studies of the cell cycle and regulated formation and activation of the replication fork have important implications for the understanding of regulation of cell proliferation. DNA replication cycles are also a target of regulation during development and differentiation. This research is expected to provide important insights into these processes.