Peconstitution and analysis of mammalian DNA replication system in vitro.

为了了解哺乳动物细胞中DNA复制的分子机制，我们尝试了生化方法来重建和分析DNA在体外复制的几个步骤，以及遗传方法来分离和鉴定DNA温度敏感突变体。我们已经从小鼠FM3A细胞中纯化了与DNA复制相关的酶和蛋白质，如DNA聚合酶和DNA拓扑异构酶复合体，四种DNA依赖的ATPase，以及一种刺激DNA聚合酶活性的因子。解开双链DNA的步骤。我们试图检测参与DNA复制这一步的DNA解旋酶的活性，并在四种DNA依赖的ATPase之一ATPase B中发现了这种活性。研究表明，解旋酶的活性依赖于ATP的水解，该酶可以解开大约120个碱基长的DNA。在单链DNA上合成Okazaki片段的步骤。我们建立了解离和重组DNA聚合酶-α-gt；复合体的条件，并用上述技术分析了确定RNA引物大小和将引物RNA合成切换到DNA合成的机理。研究表明，DNA聚合酶与底物酶形成的复合体以及脱氧核糖核苷三磷酸的存在在上述过程中起着非常重要的作用。在单链DNA上合成DNA的步骤。我们鉴定了一种刺激DNA聚合酶活性的因子，并指出该因子的刺激是由于从引物开始合成DNA的频率增加所致。

In order to understand the molecular mechanism of DNA replication in mammalian cells, we have tried biochemical approaches to reconstitute and analyze several steps of DNA replication in vitro as well as genetic approaches to isolate and characterize DNA temperature-sensitive mutants. We have purified from mouse FM3A cells enzymes and proteins related to DNA replication such as DNA polymerase <alpha> -primase complex, DNA topoisomerases <I> and <II> , four DNA-dependent ATPases, and a factor stimulating DNA polymerase <alpha> activity.1. The step to unwind double-stranded DNA.We have tried to detect DNA helicase activity which is thought to participate in this step of DNA replication, and found the activity in one of the four DNA-de-pendent ATPases, ATPase B. It has been revealed that the helicase activity is dependent on the hydrolysis of ATP, and the enzyme can unwind up to about 120 base-long DNA.2. The step to synthesize Okazaki fragments on single-stranded DNA.We have established the conditions to dissociate and reconstitute the DNA poymerase <alpha> complex and analyzed by using the above technique the mechanism to determine the size of RNA primers and to switch primer RNA synthesis to DNA synthesis. It has been revealed that the complex formation between DNA polymerase <alpha> and primase and the existence of deoxyribonucleoside triphosphates play very important roles in the above processes.3. The step to synthesize DNA on single-stranded DNA.We have characterized a factor stimulating DNA polymerase <alpha> activity and indicated that the stimulation by the factor is due to the increase in the initiation frequency of DNA synthesis from the primers.