Silver L L, Nossal N G
J Biol Chem. 1982 Oct 10;257(19):11696-705.
An activity dependent on an intact bacteriophage T4 gene 61 is required, along with the T4 gene 41 protein, for the synthesis of ribonucleotide primers in an in vitro T4 DNA replication system. In this paper, we present a method for purification of the protein catalyzing this gene 61-dependent activity based on an assay for primer-dependent DNA synthesis by the T4 DNA replication proteins. The T4 gene 32 helix-destabilizing protein influences the chromatographic behavior of 61 protein. The purification of 61 protein to near homogeneity by the scheme presented requires the presence of 32 protein in crude extracts. The isolated 61 protein is basic, with a molecular weight of 44,000, and is active as a monomer. Ribonucleotide primer synthesis shows a linear dependence on 61 protein concentration, but a sigmoidal dependence on 41 protein concentration. The dependence on 41 protein concentration is linear, however, if the 41 protein is first "activated" by incubation at high concentration in the presence of rGTP. Using this "activated" 41 protein and purified 61 protein, we show a stoichiometric relationship between the two proteins in the priming reaction consistent with the existence of a priming complex comprising an oligomer of 41 protein and a 61 protein monomer.
在体外T4 DNA复制系统中，合成核糖核苷酸引物需要一种依赖于完整噬菌体T4基因61的活性，以及T4基因41蛋白。在本文中，我们基于T4 DNA复制蛋白对引物依赖性DNA合成的测定，提出了一种纯化催化这种依赖基因61活性的蛋白质的方法。T4基因32解旋稳定蛋白会影响61蛋白的色谱行为。按照所提出的方案将61蛋白纯化至接近均一性，需要粗提物中存在32蛋白。分离得到的61蛋白呈碱性，分子量为44,000，以单体形式具有活性。核糖核苷酸引物合成对61蛋白浓度呈线性依赖，但对41蛋白浓度呈S形依赖。然而，如果首先在rGTP存在下将41蛋白在高浓度下孵育使其“激活”，那么对41蛋白浓度的依赖就是线性的。使用这种“激活的”41蛋白和纯化的61蛋白，我们在引发反应中显示出这两种蛋白之间的化学计量关系，这与存在由41蛋白寡聚体和61蛋白单体组成的引发复合物相一致。
