CYCLIN CONTROL OF DNA REPLICATION

DNA 复制的细胞周期控制

• 批准号: 6019209
• 负责人: PETER Kent JACKSON
• 金额: $ 26.54万
• 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 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6019209
• 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.

细胞周期蛋白和细胞周期蛋白复合物的瞬时积累和激活 细胞周期蛋白依赖性激酶 (Cdks) 触发进入细胞的承诺 循环并启动真核生物中染色体 DNA 的复制。在 在脊椎动物中，细胞周期蛋白 E/Cdk2 激酶已被研究其中枢 61. 这些活动包括控制 G1 和 S 期特异性转录、肿瘤抑制因子的调节、 以及染色体DNA复制的启动。 来自非洲爪蟾的两栖动物卵为研究提供了独特的模型 DNA 复制的细胞调节和要求。使用精子 非洲爪蟾卵的细胞核和无细胞提取物，复杂的反应 核组装、染色质组装和 DNA 复制继续进行 在没有蛋白质合成的情况下有效。尽管如此， DNA 复制过程中对细胞周期蛋白依赖性激酶的需求 将复制限制为每个细胞周期一轮的控制是忠实的 在这些摘录中进行了概括。杰克逊博士发明了一种非洲爪蟾提取物 依赖于外源添加的细胞周期蛋白启动的系统 DNA 复制。该系统将用于识别目标 细胞周期蛋白依赖性激酶和复制机制。研究 所描述的建议是 (1) 确定细胞周期蛋白 E 激酶的结构域 促进 DNA 复制起始所需； (2) 识别 DNA 复制的细胞周期蛋白 E 相关效应子； (3) 研究如何 细胞周期蛋白 E 激酶控制启动机制的一个组成部分： 由最近克隆的 S 的脊椎动物同源物编码的蛋白质。 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。这些方面的缺陷 调节因子和细胞周期蛋白本身涉及多种 癌症。目前，我们对 Cdk 的调控只有一个模糊的轮廓， DNA复制的时间和保真度的控制。洞察 DNA 复制的正常控制对于理解 DNA 复制如何进行非常重要 患病细胞中的复制发生改变。通过专注于识别 调节机制和复制机制的组成部分，我们可以 寻找肿瘤细胞化疗控制的新靶点。 最后，了解限制染色体的机制 每个细胞周期准确复制一次将产生影响 了解衰老、肿瘤进展、突变的产生 导致出生缺陷和代谢性疾病的原因及机制 化疗后耐药基因的扩增。