Gottlieb J, Marcy A I, Coen D M, Challberg M D
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
J Virol. 1990 Dec;64(12):5976-87. doi: 10.1128/JVI.64.12.5976-5987.1990.
Genetic experiments have shown that the products of the herpes simplex virus type 1 (HSV-1) DNA polymerase (UL30) and UL42 genes are both required for viral DNA replication, and a number of studies have suggested that these two proteins specifically interact. We have confirmed and extended these findings. The viral DNA polymerase from HSV-1-infected cells has been purified as a complex containing equimolar quantities of the UL30 (Pol, the catalytic subunit) and UL42 polypeptides. Sedimentation and gel filtration analyses of this complex are consistent with the idea that the complex consists of a heterodimer of Pol and UL42. A complex with identical physical and functional properties was also purified from insect cells coinfected with recombinant baculoviruses expressing the two polypeptides. Therefore, the formation of the Pol-UL42 complex does not require the participation of any other HSV-encoded protein. We have compared the catalytic properties of the Pol-UL42 complex with those of the isolated subunits of the enzyme purified from recombinant baculovirus-infected insect cells. The specific activity of the catalytic subunit alone was nearly identical to that of the complex when assayed on activated DNA. When assayed on a defined template such as singly primed M13 DNA, however, the combination of Pol and UL42 utilized fewer primers and formed larger products than Pol alone. Template challenge experiments demonstrated that the Pol-UL42 complex was more highly processive than Pol alone. Our data are consistent with the idea that the UL42 polypeptide is an accessory subunit of the DNA polymerase that acts to increase the processivity of polymerization.
遗传学实验表明，单纯疱疹病毒1型（HSV-1）DNA聚合酶（UL30）和UL42基因的产物都是病毒DNA复制所必需的，并且多项研究表明这两种蛋白质会特异性相互作用。我们已证实并扩展了这些发现。来自HSV-1感染细胞的病毒DNA聚合酶已被纯化，形成一种复合物，其中含有等摩尔量的UL30（Pol，催化亚基）和UL42多肽。对该复合物的沉降和凝胶过滤分析与该复合物由Pol和UL42的异二聚体组成的观点一致。从共感染表达这两种多肽的重组杆状病毒的昆虫细胞中也纯化出了具有相同物理和功能特性的复合物。因此，Pol-UL42复合物的形成不需要任何其他HSV编码蛋白的参与。我们已将Pol-UL42复合物的催化特性与从重组杆状病毒感染的昆虫细胞中纯化的该酶的分离亚基的催化特性进行了比较。在活化DNA上进行测定时，单独的催化亚基的比活性与复合物的比活性几乎相同。然而，在诸如单引物M13 DNA等确定的模板上进行测定时，与单独的Pol相比，Pol和UL42的组合使用的引物更少，形成的产物更大。模板挑战实验表明，Pol-UL42复合物比单独的Pol具有更高的持续合成能力。我们的数据与UL42多肽是DNA聚合酶的辅助亚基这一观点一致，该亚基的作用是提高聚合反应的持续合成能力。
