CONTROL OF DNA REPLICATION AND CELL DIVISION

DNA 复制和细胞分裂的控制

• 批准号: 2095911
• 负责人: TERESA S WANG
• 金额: $ 29.82万
• 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/2095911
• 关键词: DNA directed DNA polymerase; DNA replication origin; Escherichia coli; Saccharomyces; antiserum; cell cycle; cell cycle proteins; cell growth regulation; chimeric proteins; fungal genetics; gene complementation; gene expression; genetic transcription; immunoprecipitation; laboratory rabbit; lethal genes; polymerase chain reaction; posttranslational modifications; transcription factor; 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.

癌症治疗设计的先决条件是了解细胞生长 控制。 该提案的长期目标是了解 控制这两个顺序且相互依赖的分子机制 细胞周期事件，即 DNA 复制和有丝分裂。 研究 此处概述使用裂殖酵母 S. pombe 作为模型系统并解决 调节 DNA 复制蛋白表达的机制：如果 表达改变会对细胞分裂和细胞产生什么影响 增长是。 研究的初始阶段是获取试剂和 背景信息。 实现这些目标的实验是：（1） 粟酒裂殖酵母 DNA 聚合酶 a 基因的分离和表征 催化多肽及其p7O亚基，以及DNA聚合酶基因 δ大亚基及其辅助蛋白PCNA。 (2) 生产 针对这两种粟酒裂殖酵母 DNA 聚合酶及其相关抗体的抗体 蛋白质。 (3)裂殖酵母DNA基因表达的研究 聚合酶及其相关蛋白在转录和 两个 DNA 的翻译水平和翻译后修饰 细胞周期中的聚合酶。 裂殖酵母是研究最深入的有丝分裂控制系统 真核生物，但对其 DNA 调控机制知之甚少 复制以及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，但是 翻译后 pol α 在细胞内组成型表达 周期，通过关键的有丝分裂以细胞周期依赖性方式磷酸化 调节器 p34(cdc2) 相关激酶。 在初始阶段之后 研究中，我们将：（1）识别细胞周期依赖性翻译后 修饰残基并对这些残基进行诱变； (2) 检测表型 这些突变体在细胞周期中通过破坏裂殖酵母基因而产生 然后用诱变基因进行补充； (3)构建 用于分离抑制子的条件致死突变体。 这些研究 将提供有关监管机制的新颖信息 细胞生长过程中的前导链和滞后链 DNA 聚合酶，并为 研究细胞如何调节 DNA 复制顺序和细胞 分配。 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。