MECHANISM OF DNA REPLICATION IN EUCARYOTES--YEAST AS A MODEL SYSTEM

真核生物DNA复制机制——以酵母为模型系统

• 批准号: 3918718
• 负责人: A SUGINO
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3918718
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA topoisomerases; antibody; enzyme mechanism; eukaryote; fungal genetics; gene expression; genetic library; molecular cloning; nucleic acid sequence; pancreatic ribonuclease; plasmids; temperature sensitive mutant; tissue /cell culture; yeasts

An in vitro DNA replication system using yeast 2-Mum and ARS (autonomously replicating sequences) plasmid DNAs, developed in this laboratory, has been used to investigate the mechanism of DNA replication in yeast. To identify and purify enzymes and components required for yeast chromosomal DNA replication, the crude extract system has been fractionated and reconstituted with the help of several temperature-sensitive chromosomal DNA replication mutants. To aid in overproducing and purifying such DNA replication proteins, the DBF1 and 2 genes (which are required for the elongation step of DNA replication) and the TS26 gene (required for the initiation of DNA replication) have been cloned, their nucleotide sequences determined, antibodies to them raised and their regulation studied. lt was found that the DBF2 protein is a serine/threonine-specific protein kinase, suggesting that protein phosphorylation regulates elongation of DNA synthesis in yeast. Finally, yeast DNA polymerases I, II, and III (The last being newly discovered in yeast crude extracts) have been purified to homogeneity for the first time and studied extensively. Using inhibitors and antibodies against each purified DNA polymerase, it has been established that each polymerase is unique and has a different function in yeast cells. In order to study these functions, the molecular cloning of DNA polymerases I, II and III is underway using either antibodies or the amino acid sequences Df oligopeptides generated from the purified polymerases. In the meantime, mammalian PCNA/cycling, which is under cell-cycle control and is a subunit of DNA polymerase delta, has been shown to stimulate the yeast DNA polymerase III reaction, suggesting that yeast DNA polymerase III is a homolog of mammalian DNA polymerase delta.

酵母2-Mum和ARS体外DNA复制系统的建立 （自主复制序列）质粒DNA， 这个实验室，已经被用来研究DNA的机制， 酵母中的复制 为了鉴定和纯化酶， 酵母染色体DNA复制所需的成分， 将粗提物系统分馏并用以下物质重构 几种温度敏感染色体DNA的帮助 复制突变体 为了帮助过度生产和净化这些 DNA复制蛋白，DBF 1和2基因（这是必需的 用于DNA复制的延伸步骤）和TS 26基因 （DNA复制起始所需的）已经被克隆， 它们的核苷酸序列被确定， 研究了它们的规律。 发现DBF 2蛋白 是丝氨酸/苏氨酸特异性蛋白激酶，这表明， 蛋白质磷酸化调节DNA合成的延长， 酵母 最后，酵母DNA聚合酶I、II和III（最后一种是新的 在酵母粗提物中发现）已被纯化， 第一次被广泛研究。 使用 针对每种纯化的DNA聚合酶的抑制剂和抗体， 已经确定每种聚合酶是独特的， 在酵母细胞中的不同功能。 为了研究这些 DNA聚合酶I、II和III的分子克隆 正在使用抗体或氨基酸序列Df进行 从纯化的聚合酶产生的寡肽。 在 与此同时，哺乳动物细胞增殖细胞核抗原/细胞周期，这是在细胞周期控制 是DNA聚合酶δ的一个亚基， 刺激酵母DNA聚合酶III反应，表明 酵母DNA聚合酶III是哺乳动物DNA聚合酶的同系物 δ的