ENZYMATIC MECHANISMS OF DNA REPLICATION

描述：该提案的目标是通过以下方式了解该机制： 其中DNA复制起点被转化为复制叉， 分叉处的蛋白质是如何组织的， 领先股和落后股。 基本假设，虽然 有一种说法是，存在一个超级集合体， 蛋白质在复制叉，它的功能就像一台机器， 活动的部分是蛋白质。 不太确定的是， 以协调一致的方式改变构象的在起源处组装的复合物 以影响分叉进程的每个阶段，或者如果存在多个较小的 每台机器都被分配了不同的特定任务， 与叉有关。 要解决如此复杂的问题， 将其分解为部分反应。 在即将到来的拨款期内， 研究人员将深入研究DNA 2解旋酶和DNA聚合酶 蛋白质/蛋白质相互作用的动力学， 这些关键组成部分。 他们将使用酵母作为模型系统， 结合遗传学和生物化学的方法。 在过去的授权期间，研究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， 与FEN-1相互作用，FEN-1是一种参与冈崎片段加工的蛋白质， SV 40体外DNA复制。 对DNA 2的进一步研究的目标是 确定DNA 2解旋酶是否在细胞的成熟中起作用， 滞后链在酵母细胞，如果是这样，它是否有催化作用或 建筑/结构的作用。 不言而喻，要理解组装、激活和 为了了解复制叉的运动，我们必须了解DNA聚合酶。 这项资助支持了许多酵母DNA聚合酶的研究， 年 最近，调查人员把注意力集中在了警察身上。 他们有 使用定点突变分析C-末端的一半， 催化核心多肽，可能在DNA复制中具有双重作用 并感知DNA损伤。 调查人员将继续确定 蛋白质/蛋白质接触的聚合酶全酶，并确定 各子单位的作用。 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， 复制的起点和分叉，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 在此资助下生产的突变体和克隆体。

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.