ENZYMATIC MECHANISMS OF DNA REPLICATION

DNA 复制的酶促机制

• 批准号: 6603184
• 负责人: Judith L CAMPBELL
• 金额: $ 10.44万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-01 至 2003-04-08
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6603184
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA replication origin; bacteriophage T7; crosslink; dimer; enzyme mechanism; enzyme structure; enzyme substrate; helicase; mass spectrometry; proliferating cell nuclear antigen; protein structure; site directed mutagenesis; yeast two hybrid system

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.

描述：该提案的目标是通过以下方式理解该机制： 其DNA复制起点被转换成复制叉并且 叉子上的蛋白质如何组织以协调合成 领先和落后的股。 基本假设，虽然 似乎有待证明的是，存在一个超级集合 复制叉上的蛋白质，它的功能就像一台机器 运动部件是蛋白质。 不太确定的是是否有一个单一的 在起源处组装的复合物，以协调一致的方式改变构象 影响分叉进展的每个阶段，或者如果有许多较小的阶段 每台机器都分配了不同的特定任务，并且只是暂时的 与叉子相关联。 要解决如此复杂的问题需要 将其分解为部分反应。 在接下来的授予期内 研究人员将深入研究 Dna2 解旋酶和 DNA 聚合酶 epsilon，重点关注蛋白质/蛋白质相互作用的动态 这些关键部件。 他们将使用酵母作为模型系统来允许 结合遗传和生化方法。 在过去的授权期间，研究人员描述了一种解旋酶 对于染色体 DNA 复制至关重要。 结果发现，它 与 FEN-1 相互作用，FEN-1 是一种参与冈崎片段加工的蛋白质 SV40体外DNA复制。 对 Dna2 的进一步研究的目标是 确定 Dna2 解旋酶是否在成熟过程中发挥作用 酵母细胞中的滞后链，如果是的话，它是否具有催化作用或 建筑/结构作用。 不言而喻，要理解组装、激活和 复制叉的运动，必须了解DNA聚合酶。 这笔资助支持了许多酵母 DNA 聚合酶的研究 年。 最近，研究人员将注意力集中在 pol epsilon 上。 他们有 在对 C 端一半的分析中使用了定点诱变 催化核心多肽，可能在 DNA 复制中具有双重作用 并检测 DNA 损伤。 调查人员将继续确定 pol epsilon 全酶中的蛋白质/蛋白质接触并确定 各个亚基的作用。 他们将利用染色质交联来研究 三种 DNA 聚合酶之间相互作用的动态 复制起点和分叉，最终要解决哪个问题 聚合酶复制哪条链，利用所有聚合酶 在这笔赠款下产生的突变体和克隆。