MECHANISM OF DNA REPLICATION IN EUCARYOTES--I 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: 4693266
• 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/4693266
• 关键词: DNA directed DNA polymerase; antibody; deoxyribonuclease I; enzyme structure; eukaryote; gene expression; genetic library; genetic models; model design /development; molecular cloning; nucleic acid sequence; plasmids; restriction endonucleases; temperature sensitive mutant; tissue /cell culture; yeasts

An in vitro DNA replication system using yeast 2-Mum and ARS plasmid DNA has been developed as a model to investigate the mechanism of DNA replication in eucaryotes. To elucidate the mechanisms of initiation, elongation and termination of DNA replication, this system has been further characterized. Recent biochemical fractionation and reconstitution experiments permitted the purification and characterization of yeast DNA primase, (60,000 daltons), single-stranded DNA binding protein, (38,000 daltons), DNA-dependent ATPase III (63,000 daltons) and DNA polymerase I (140,000 daltons). Furthermore, RNase Hs (21,000, 48,000 and 68,000 daltons) and two other single-stranded DNA binding proteins (14,000 and 20,000 daltons) which might participate in DNA replication have been purified to homogeneity by following their enzymatic activities. To prove that these proteins are required for yeast DNA replication, antibodies have been raised against each and their genes have been identified and cloned from Gammagt11 yeast genomic library using the antibodies. Currently, their nucleotide sequences are being determined. In order to permit identification and isolation of other DNA replication proteins, new temperature-sensitive DNA replication mutants of yeast have been isolated and characterized genetically; some of these mutant genes have been identified and cloned from the yeast genomic library.

酵母2-Mum和ARS质粒DNA体外复制体系的建立 已被开发为研究DNA机制的模型 真核生物中的复制 为了阐明引发的机制， 延长和终止DNA复制，该系统已被进一步 表征了 最近的生化分离和重组 实验允许酵母DNA的纯化和表征， 引物酶，（60，000道尔顿），单链DNA结合蛋白，（38，000道尔顿 道尔顿）、DNA依赖性ATP酶III（63，000道尔顿）和DNA聚合酶I (140，000道尔顿）。 此外，RNase Hs（21，000、48，000和68，000 道尔顿）和另外两种单链DNA结合蛋白（14，000和 20，000道尔顿）可能参与DNA复制， 通过跟踪它们的酶活性将其纯化至均一。 证明 这些蛋白质是酵母DNA复制所必需的，抗体具有 它们的基因已经被鉴定和克隆 从Gammagt 11酵母基因组文库中获得。 目前， 它们的核苷酸序列正在测定中。 为了允许 其他DNA复制蛋白的鉴定和分离，新 已经分离出酵母的温度敏感性DNA复制突变体 并在基因上进行了表征;其中一些突变基因已经被 从酵母基因组文库中鉴定并克隆。