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

利用酵母2-MUM和ARS质粒DNA的体外DNA复制系统 已被开发为研究DNA机制的模型 真核细胞中的复制。为了阐明启动的机制， 延长和终止DNA复制，这一系统进一步 特色化的。最新的生化分离与重组 实验允许酵母DNA的提纯和鉴定 Primase(60,000道尔顿)，单链DNA结合蛋白(38,000 DNA依赖的ATPase III(63,000道尔顿)和DNA聚合酶I (14万道尔顿)。此外，RNase HS(21,000、48,000和68,000 和另外两个单链DNA结合蛋白(14,000和 20,000道尔顿)中可能参与DNA复制的 通过跟踪它们的酶活性来纯化到均一。为了证明 这些蛋白质是酵母DNA复制所必需的，抗体 它们的基因已经被鉴定和克隆 从Gammagt11酵母基因组文库中提取抗体。目前， 它们的核苷酸序列正在确定中。为了允许 其他DNA复制蛋白的鉴定和分离，新 分离到温度敏感的酵母DNA复制突变体 其中一些突变基因已经被 从酵母基因组文库中鉴定和克隆。

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.