ENZYMATIC MECHANISMS OF DNA REPLICATION

描述：提案的目标是通过以下方式了解该机制 其中DNA复制的起点被转换为复制叉，并且 叉子上的蛋白质是如何组织起来的，以协调 领先的和落后的一股。潜在的假设，虽然 可信的，有待证明的是，有一个超级集会 蛋白质在复制叉上，它的功能就像一台机器，它的 运动部分是蛋白质。不太确定的是，是否有一支 在原点组装的以协调方式改变构象的复合体 以实现分叉进程的每个阶段，或者如果有一些较小的 每台机器都分配了不同的特定任务，并且仅是暂时的 与叉子相关联。要解决如此复杂的问题，需要 将其分为部分反应。在接下来的授权期内， 研究人员将深入研究DNA2解旋酶和DNA聚合酶 Epsilon，重点研究了蛋白质/蛋白质相互作用的动力学。 这些关键组件。他们将使用酵母作为模型系统，以允许 遗传和生化相结合的方法。 在过去的授权期内，调查人员描述了一种解旋酶 对染色体DNA复制至关重要。人们发现，它 与Fen-1相互作用，Fen-1是一种参与冈崎片段处理的蛋白质 SV40在体外进行DNA复制。对DNA2的进一步研究的目标是 确定DNA2解旋酶是否在细胞成熟过程中起作用 酵母细胞中的滞后链，如果是这样，它是否具有催化作用或 建筑/结构角色。 不用说，要理解组装、激活和 复制叉子的移动，必须了解DNA聚合酶。 这笔赠款支持了许多年来对酵母DNA聚合酶的研究 好几年了。最近，调查人员将重点放在了Polepsilon身上。他们有 用定点突变技术分析猪细小病毒的C-末端半部分 催化核心多肽，可能在DNA复制中具有双重作用 并感觉到DNA的损伤。调查人员将继续确定 Polepsilon全酶中的蛋白质/蛋白质接触并确定 个别亚基的作用。他们将使用染色质交联来研究 三种DNA聚合酶之间相互作用的动力学 复制起源和分支，最终要解决的问题是 聚合酶复制哪条链，利用所有的聚合酶 在这项资助下产生的突变体和克隆人。

DESCRIPTION: The goal of the proposal 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, remain to be proved, is that there is a super assembly of proteins at the replication fork and that it functions like a machine whose moving parts are proteins. Wha is less certain is whether there is a single complex assembled 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 investigator will study in depth the Dna2 helicase and DNA polymerase epsilon, with a focus on the dynamics of protein/protein interactions of these key components. They would use yeast as model system to allow a combined genetic and biochemical approach. In the past granting period, the investigators described a helicase essential for chromosomal DNA replication. It was discovered that it interacted with FEN-1, a protein involved in Okazaki fragment processing in SV40 in vitro DNA replication. Further studies of Dna2 have the goal of determining whether the Dna2 helicase plays a role in maturation of the lagging strand in yeast cells and if so, does it have a catalytic role or an architectural/structural role. It should go without saying that to understand the assembly, activation, and movement of the replication fork, one must understand the DNA polymerase. This grant has supported studies of the yeast DNA polymerases for many years. Recently the investigators have focused on pol epsilon. They have used site-directed mutagenesis in an analysis of the C-terminal half of the catalytic core polypeptide, which may have a dual role in DNA replication and sensing DNA damage. The investigators will continue to determine the protein/protein contacts in the pol epsilon holoenzyme and to determine the role of individual subunits. They will use chromatin crosslinking to study the dynamics of interactions between the three DNA polymerases at replication origins and forks, ultimately to get at the question of which polymerase copie which strand, taking advantage of all the polymerase mutants and clones produced under this grant.