CONTROL OF DNA REPLICATION AND CELL DIVISION

DNA 复制和细胞分裂的控制

• 批准号: 3198970
• 负责人: TERESA S WANG
• 金额: $ 27.93万
• 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/3198970
• 关键词: DNA directed DNA polymerase; DNA replication origin; Escherichia coli; Saccharomyces; antiserum; 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细胞生长的知识 控制。 该提议的长期目标是了解 控制两个顺序和相互依赖的分子机制 细胞周期的事件，即DNA复制和有丝分裂。 研究 此处概述使用裂变酵母，S。Pombe作为模型系统和地址 调节DNA复制蛋白表达的机制：if 表达会改变对细胞分裂和细胞的影响 增长是。 该研究的初始阶段是获得试剂和 背景信息。 实现这些目标的实验是：（1） S. pombe 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聚合酶的基因 Delta大亚基及其辅助蛋白PCNA。 （2）生产 针对这两种S. pombe DNA聚合酶及其相关的抗体 蛋白质。 （3）研究裂变酵母DNA的基因表达 聚合酶及其相关蛋白在转录和 翻译水平和两个DNA的翻译后修饰 在细胞周期中聚合酶。 裂变酵母是研究最佳的有丝分裂控制系统 真核生物，但对其DNA的调节机制知之甚少 复制以及复制与有丝分裂的相互依存关系。 在哺乳动物细胞中，滞后链DNA聚合酶，但 在细胞期间组成型表达后倾斜度alpha 循环，通过关键有丝分裂以细胞周期依赖性方式磷酸化 调节剂p34（CDC2）相关激酶。 在初始阶段之后 研究，我们将：（1）在翻译后识别细胞周期依赖性 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裂变酵母基因的破坏 然后与诱变基因互补。 （3）结构 有条件的致命突变体，用于分离抑制器。 这些研究 将提供有关调节机制的新颖信息 细胞生长过程中的铅和滞后DNA聚合酶，并铺平 研究细胞如何调节DNA复制和细胞的顺序 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提供有价值的推断 在癌症疗法中。