CYCLIN CONTROL OF DNA REPLICATION

DNA 复制的细胞周期控制

• 批准号: 6463979
• 负责人: PETER Kent JACKSON
• 金额: $ 8.85万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2002-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6463979
• 关键词: DNA replication; SDS polyacrylamide gel electrophoresis; Schizosaccharomyces pombe; Xenopus oocyte; antiserum; cell cycle; cell cycle proteins; cyclin dependent kinase; cyclins; enzyme activity; high performance liquid chromatography; immunoaffinity chromatography; immunoprecipitation; laboratory mouse; laboratory rabbit; molecular cloning; phosphorylation; posttranslational modifications; protein purification; protein sequence; protein structure function; tissue /cell culture

The transient accumulation and activation of complexes of cyclins and cyclin-dependent kinases (Cdks) triggers the commitment to enter the cell cycle and to initiate chromosomal DNA replication in eukaryotes. In vertebrates, the cyclin E/Cdk2 kinase has been studied for its central role in promoting activities in 61. These activities include control of G1- and S-phase specific transcription, regulation of tumor suppressors, and the initiation of chromosomal DNA replication. The amphibian egg from Xenopus laevis provides a unique model for studying the cellular regulation and requirements for DNA replication. Using sperm nuclei and cell-free extracts from Xenopus eggs, the complex reactions of nuclear assembly, chromatin assembly, and DNA replication proceed efficiently in the absence of protein synthesis. Nonetheless, the requirement for cyclin-dependent kinases in DNA replication and the control that limits replication to one round per cell cycle are faithfully recapitulated in these extracts. Dr. Jackson has devised a Xenopus extract system that is dependent on exogenously added cyclins for the initiation of DNA replication. This system will be used to identify targets of the cyclin-dependent kinases and mechanisms of replication. The research described proposes to (1) determine the domains of the cyclin E kinase required for promoting the initiation of DNA replication; (2) identify cyclin E-associated effectors of DNA replication; and (3) to study how the cyclin E kinase controls a component of the initiation machinery: the protein encoded by a recently cloned vertebrate homologue of the S. cerevisiae CDC6 gene. The regulatory proteins that modulate G1 cyclin/Cdk kinases are important for controlling cellular growth and senescence. Defects in these regulators and cyclins themselves are implicated in a wide variety of cancers. At this time, we have only a faint outline of Cdk regulation and the control of timing and fidelity of DNA replication. Insight into the normal control of DNA replication is important to understanding how replication is altered in diseased cells. By focusing on identifying components of the regulatory mechanism and replication machinery, we may find new targets for chemotherapeutic control of neoplastic cells. Finally, understanding the mechanisms that constrain chromosomes to replicate accurately once per cell cycle will have implications for understanding aging, tumor progression, the generation of mutations that cause birth defects and metabolic disease, and the mechanism of amplification of drug resistance genes following chemotherapy.

细胞周期蛋白和复合物的瞬时积累和激活 Cyclin依赖性激酶（CDKS）触发进入细胞的承诺 循环并启动真核生物中的染色体DNA复制。在 脊椎动物，细胞周期蛋白E/CDK2激酶已被研究以其中央 在61中促进活动中的作用。这些活动包括控制 G1和S期特异性转录，调节肿瘤抑制剂， 以及染色体DNA复制的启动。 来自Xenopus laevis的两栖鸡蛋提供了一个独特的模型 细胞调节和DNA复制的要求。使用精子 来自爪蟾卵的细胞核和无细胞提取物， 核装配，染色质组件和DNA复制继续进行 在没有蛋白质合成的情况下有效地有效。尽管如此， 在DNA复制中对细胞周期蛋白依赖性激酶的需求和 控制将复制限制为每个单元周期的一个回合是忠实的 在这些提取物中概括。杰克逊博士设计了一种武士提取物 取决于启动的外源添加细胞周期蛋白的系统 DNA复制。该系统将用于识别 依赖细胞周期蛋白的激酶和复制机制。研究 所描述的提议（1）确定细胞周期蛋白E激酶的结构域 促进DNA复制的启动所需； （2）识别 Cyclin E与DNA复制的效应子； （3）研究如何 细胞周期蛋白E激酶控制起始机械的组成部分： 蛋白质由S.最近克隆的脊椎动物同源物编码 酿酒酵母CDC6基因。 调节G1细胞周期蛋白/CDK激酶的调节蛋白很重要 用于控制细胞生长和衰老。这些缺陷 调节剂和细胞周期蛋白本身与多种多样 癌症。目前，我们只有一个微弱的CDK调节概述和 DNA复制的时间和忠诚度的控制。深入了解 对DNA复制的正常控制对于理解如何 患病细胞的复制改变。通过专注于识别 监管机制和复制机制的组件，我们可能会 找到用于肿瘤细胞化学治疗控制的新靶标。 最后，了解将染色体限制为 每个细胞周期准确复制一次将对 了解衰老，肿瘤进展，突变产生 导致先天缺陷和代谢疾病，以及 化疗后耐药基因的扩增。