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: 3965287
• 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/3965287
• 关键词: DNA topoisomerases; adenosinetriphosphatase; antibody; eukaryote; gene expression; genetic library; genetic models; molecular cloning; nucleic acid sequence; pancreatic ribonuclease; plasmids; temperature sensitive mutant; tissue /cell culture; yeasts

An in vitro DNA replication system of yeast 2-Mum and ARS1 plasmid DNAs has been used as a model to investigate the mechanism of DNA replication in eucaryotes. To identify various enzymes and protein factors required for DNA replication, the system has been further fractionated and reconstituted by using separated fractions. From these experiments, two additional proteins have been identified and purified. These are newly identified single-stranded DNA-dependent ATPase (ATPase III) and DNA topoisomerase II and have been extensively studied in this year. The ATPase III has additional enzymatic activity which unwinds double-stranded DNA utilizing energy of ATP hydrolysis (helicase activity). Furthermore, RNase Hs and single-stranded DNA binding proteins which might participate in DNA replication have been purified to homogeneity by following their enzymatic activities. To provide that these proteins are required for yeast DNA replication, antibodies have been raised aginst each and their genes have been identified and cloned from lambda gt11 expression yeast genomic library using the antibodies. Their nucleotide sequences have been determined and the genes have been mapped on the chromosomes. Finally, the essentiality of each gene has been tested by gene disruption and one of single-stranded DNA binding proteins (20k dalton) is found to be required for yeast cell viability. In order to permit identification and isolation of other DNA replication proteins, new temperature-sensitive DNA replication mutants of yeast have been isolated, characterized genetically, some of these mutant genes have been cloned and their nucleotide sequences have been determined. Currently, to ease the purification of their gene products overproduction of the gene products is being carried out.

酵母2-Mum和ARS1质粒DNA的体外DNA复制系统 被用来作为研究DNA复制机制的模型 真核生物。确定各种酶和蛋白质因子所需的 DNA复制，系统被进一步分离和重组 通过使用分开的分数。从这些实验中，又增加了两个 蛋白质已经被鉴定和提纯。这些都是新鉴定的 单链DNA依赖的ATPase III和DNA拓扑异构酶II 并在今年进行了广泛的研究。ATPase III拥有 利用额外的酶活性解开双链DNA ATP水解能(解旋酶活性)。此外，RNase HS和 可能参与DNA的单链DNA结合蛋白 通过遵循它们的酶促反应，复制被纯化到同质性 活动。为了提供酵母DNA所需的这些蛋白质 复制，针对每个人的抗体都被提高了，它们的基因 从表达lambda gt11的酵母基因组中鉴定和克隆 使用抗体的文库。它们的核苷酸序列已经被 并且基因已经被定位在染色体上。最后， 每个基因的重要性都通过基因破坏得到了检验，其中一个 发现需要单链DNA结合蛋白(20k道尔顿) 对于酵母细胞的活力。为了允许识别和隔离 在其他DNA复制蛋白中，新的温度敏感型DNA 酵母的复制突变体已经被分离出来，并对其进行了遗传特征分析， 其中一些突变基因已经被克隆，它们的核苷酸序列 已经确定了。目前，为了简化他们的基因纯化 目前正在进行基因产品的生产过剩。