CONTROL OF DNA REPLICATION AND CELL DIVISION

DNA 复制和细胞分裂的控制

• 批准号: 3198969
• 负责人: TERESA S WANG
• 金额: $ 27.76万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3198969
• 关键词: DNA directed DNA polymerase; DNA replication origin; Escherichia coli; Saccharomyces; antiserum; cell growth regulation; chimeric proteins; fungal genetics; gene complementation; gene expression; genetic transcription; immunoprecipitation; laboratory rabbit; lethal genes; polymerase chain reaction; posttranslational modifications; transposon /insertion element

Prerequisite for design of cancer therapy is the knowledge of cell growth control. The long term goal of this proposal is to understand the molecular mechanisms that control the two sequential and interdependent events of the cell cycle, i.e. DNA replication and mitosis. The research outlined here uses fission yeast, S. pombe, as a model system and addresses the mechanisms regulating the expression of DNA replicative proteins: if expression is altered what will the effects on cell division and cell growth be. The initial phase of the study is to obtain reagents and background information. Experiments to achieve these objectives are: (1) Isolation and characterization of the genes of S. pombe DNA polymerase a catalytic polypeptide and its p7O subunit, and the genes of DNA polymerase delta large subunit and its auxiliary protein PCNA. (2) Production of antibodies against these two S. pombe DNA polymerases and their associated proteins. (3) Investigation of gene expression of fission yeast DNA polymerases and their associated proteins at the transcriptional and translational levels, and posttranslational modification of the two DNA polymerases during the cell cycle. Fission yeast is the best studied mitotic control system among eukaryotes, but little is known about its regulatory mechanisms of DNA replication and the interdependent relationship of replication and mitosis. In mammalian cells, the lagging strand DNA polymerase, but postranslationally pol alpha, is constitutively expressed during the cell cycle, phosphorylated in a cell cycle-dependent manner by the key mitotic regulator p34(cdc2) associated kinase. After the initial phase of the study, we will: (1) identify the cell cycle-dependent posttranslationally modified residues and mutagenize these residues; (2) test the phenotype of these mutants during the cell cycle by disruption of the fission yeast gene followed by complementation with a mutagenized gene; (3) construct conditional lethal mutants for isolation of suppressors. These studies will provide novel information about the regulatory mechanisms of the leading and lagging strand DNA polymerases during cell growth, and pave the way for studying how cells modulate the order of DNA replication and cell division. Understanding of these fundamental mechanisms regulating cell growth and division will provide valuable inferences to molecular etiology in cancer therapies.

设计癌症治疗的前提是了解细胞生长 控制 本提案的长期目标是了解 控制这两个顺序和相互依赖的分子机制 细胞周期的一系列事件，即DNA复制和有丝分裂。 研究 这里概述使用裂变酵母，S。pombe，作为一个模型系统和地址 调节DNA复制蛋白表达的机制：如果 基因表达的改变会对细胞分裂和细胞增殖产生什么影响 增长是。 研究的初始阶段是获得试剂， 背景资料。 实现这些目标的实验是：（1） 分离并鉴定了S.粟酒DNA聚合酶a 催化多肽及其p7 O亚基，以及DNA聚合酶基因 δ大亚基及其辅助蛋白PCNA。(2)生产 针对这两种S.粟酒酵母DNA聚合酶及其相关的 proteins. (3)裂殖酵母DNA基因表达的研究 聚合酶及其相关的蛋白质在转录和 翻译水平和两种DNA的翻译后修饰 聚合酶在细胞周期中。 裂变酵母是有丝分裂控制系统中研究得最好的 真核生物，但很少有人知道它的调控机制的DNA 复制和有丝分裂的相互依赖关系。 在哺乳动物细胞中，滞后链DNA聚合酶， 翻译后pol α在细胞内组成性表达， 细胞周期，磷酸化的细胞周期依赖的方式，由关键的有丝分裂 调节子p34（cdc 2）相关激酶。 在第一阶段之后， 研究中，我们将：（1）确定细胞周期依赖性posterior 修饰残基并诱变这些残基;（2）测试表型 这些突变体在细胞周期中通过分裂酵母基因的破坏 随后与诱变基因互补;（3）构建 用于分离抑制子的条件致死突变体。 这些研究 将提供新的信息的调节机制， 引导和滞后链DNA聚合酶在细胞生长过程中，并铺平了 一种研究细胞如何调节DNA复制顺序的方法， 师. 了解这些调节细胞的基本机制 生长和分裂将为分子病因学提供有价值的推论 在癌症治疗中。