DNA Helicases in DNA Replication and Repair

DNA 复制和修复中的 DNA 解旋酶

• 批准号: 9985527
• 负责人: Judith Campbell
• 金额: $ 31.33万
• 依托单位: California Institute of Technology
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9985527&HistoricalAwards=false
• 关键词: DNA; Helicases; Replication; Repair

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.

这项研究的目的是了解DNA复制起源转化为复制叉的机制，以及叉上的蛋白质是如何组织起来协调前导链和滞后链的合成的。潜在的假设，虽然看似合理，但仍有待证明，即在复制叉上有一个蛋白质的超级组装，它的功能就像一台机器，其活动部件是蛋白质。不太确定的是，在起源处是否存在一个单一的、多蛋白质的组装，以协调一致的方式改变构象，影响叉子进程的每个阶段，或者是否存在许多较小的机器，每个机器被分配了不同的特定任务，只是短暂地与叉子相关。要解决这样一个复杂的问题，需要把它分成若干部分的反应。在接下来的授予期，我们将深入研究dna - 2解旋酶及其在起始和延伸中的作用。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概括体外起始和延伸。非洲爪蟾体外复制系统忠实地模拟了在体内发生的事件，在研究起始蛋白在精子染色质上的组装顺序方面非常有用。该系统的一个重要特点是鸡蛋提取物储存了复制蛋白，能够在体外非常快速和广泛地复制。因此，它们也为研究真核细胞染色体复制分叉提供了唯一存在的体外复制系统。此前，在这个实验室中发现了酵母中DNA复制所必需的第一个DNA解旋酶。为了充分利用酵母分子生物学、遗传学和爪蟾体外复制系统的优势，克隆了与酵母解旋酶同源的爪蟾dna。非洲爪蟾提取物中DNA复制的每个阶段现在将在Xdna2p存在和不存在的情况下进行分析。在细胞周期中，在Xdna2p存在和不存在的情况下，复制装置的组装和拆卸将被遵循。DNA2缺陷的酵母突变体对x射线和MMS敏感。由于爪蟾提取物已被证明对x射线诱导损伤的修复研究有用，因此爪蟾提取物将用于研究Xdna2的分子贡献。研究细胞周期和受调控的复制叉的形成和激活对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的调控目标。这项研究有望为这些过程提供重要的见解。