CONTROL OF DNA REPLICATION AND CELL DIVISION

癌症治疗设计的先决条件是细胞生长的知识 控制力。这项建议的长期目标是理解 控制这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。这项研究 这里概述了使用分裂酵母，S.pombe，作为一个模型系统和地址 DNA复制蛋白表达的调控机制：IF 表达改变会对细胞分裂和细胞产生什么影响 增长就是。研究的初始阶段是获得试剂和 背景资料。实现这些目标的实验是：(1) 庞氏链霉菌DNA聚合酶A基因的分离与鉴定 催化多肽及其p7O亚基与DNA聚合酶基因 Delta大亚基及其辅助蛋白增殖细胞核抗原。(2)生产 抗这两种S.pombe DNA聚合酶及其相关抗体 蛋白质。(3)裂解酵母DNA基因表达的研究 转录和转录水平的聚合酶及其相关蛋白 两种DNA的翻译水平和翻译后修饰 细胞周期中的聚合酶。 分裂酵母是研究最多的有丝分裂调控系统 真核生物，但对其对DNA的调控机制知之甚少 复制以及复制和有丝分裂之间的相互依赖关系。 在哺乳动物细胞中，滞后链DNA聚合酶，但 翻译后，Pola在细胞中结构性地表达 周期，由关键的有丝分裂以依赖于细胞周期的方式磷酸化 调节因子p34(Cdc2)相关蛋白。在初始阶段之后 研究中，我们将：(1)确定翻译后细胞周期依赖性 修饰残基并诱变这些残基；(2)检测菌株的表型 这些突变体在细胞周期中通过破坏分裂酵母基因 然后与诱变基因互补；(3)构建 用于分离抑制子的条件致死突变体。这些研究 将提供有关监管机制的新信息 在细胞生长过程中领先和落后的链DNA聚合酶，并为 研究细胞如何调节DNA复制顺序和细胞的方法 组织。对这些调节细胞的基本机制的理解 生长和分裂将为分子病因学提供有价值的推论 在癌症治疗方面。

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.