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

酵母2-Mum和ARS 1质粒DNA的体外DNA复制系统， 被用作研究DNA复制机制的模型， 真核生物 为了鉴定各种酶和蛋白质因子， DNA复制，系统被进一步细分和重组 通过使用分离的馏分。 从这些实验中，另外两个 蛋白质已被鉴定和纯化。 这些是新发现的 单链DNA依赖性ATP酶（ATP酶III）和DNA拓扑异构酶II 并在今年得到了广泛的研究。 ATP酶III具有 另外的酶活性，其利用DNA解旋双链， ATP水解的能量（解旋酶活性）。 此外，RNase Hs和 单链DNA结合蛋白可能参与DNA 复制已被纯化到同质，通过以下他们的酶 活动 为了提供这些蛋白质是酵母DNA所必需的 复制，抗体已经提出了对每一个和他们的基因， 从λ gt 11表达酵母基因组中鉴定并克隆 使用抗体的文库。 它们的核苷酸序列 基因已经被定位在染色体上。 最后 每个基因的必要性已经通过基因破坏进行了测试， 发现需要单链DNA结合蛋白（20 k道尔顿） 酵母细胞的活力。 为了便于识别和隔离 其他DNA复制蛋白，新的温度敏感的DNA 酵母的复制突变体已被分离，遗传表征， 已经克隆了这些突变基因中的一些，并且它们的核苷酸序列 已经确定。 目前，为了简化他们基因的纯化 基因产物的过度生产正在进行。

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.