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

基于酵母2-MUM和ARS的DNA体外复制系统 (自主复制序列)质粒DNA，开发于 这个实验室被用来研究DNA的作用机制。 在酵母中复制。鉴定和纯化酶和 酵母染色体DNA复制所需的组件， 粗提物系统已被分馏和重组 几种温度敏感的染色体DNA的帮助 复制突变体。以帮助过度生产和净化这些 DNA复制蛋白、DBF1和2基因(这是必需的 用于DNA复制的延伸步骤)和TS26基因 (启动DNA复制所需)已被克隆， 它们的核苷酸序列被确定，对它们的抗体被提高 并对其规则进行了研究。研究发现，DBF2蛋白 是一种丝氨酸/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(最后一个是新发现的 在酵母粗提物中发现)已被提纯为 首次提出了同质性，并进行了广泛研究。vbl.使用 针对每个纯化的DNA聚合酶的抑制剂和抗体， 已经证实，每个聚合酶都是独一无二的，并且有一个 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的功能及其分子克隆 正在使用抗体或氨基酸序列df 从纯化的聚合酶中产生寡肽。在 同时，哺乳动物的增殖细胞核抗原/周期，这是在细胞周期控制下 是DNA聚合酶Delta的一个亚单位，已被证明 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聚合酶的同源物 达美航空。

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.