Hernandez T R, Lehman I R
Department of Biochemistry, Beckman Center, Stanford University School of Medicine, California 94305-5307.
J Biol Chem. 1990 Jul 5;265(19):11227-32.
The herpes simplex virus 1 (HSV-1) UL42 protein, one of seven herpes-encoded polypeptides that are required for the replication of the HSV-1 genome, is found in a 1:1 complex with the HSV-1 DNA polymerase (Crute, J. J., and Lehman, I. R. (1989) J. Biol. Chem. 264, 19266-19270). To obtain herpes DNA polymerase free of UL42 protein, we have cloned and overexpressed the Pol gene in a recombinant baculovirus vector and purified the recombinant DNA polymerase to near homogeneity. Replication of singly primed M13mp18 single-stranded DNA by the recombinant enzyme in the presence of the herpes encoded single-stranded DNA-binding protein ICP8 yields in addition to some full-length product a distribution of intermediate length products by a quasi-processive mode of deoxynucleotide polymerization. Addition of the purified UL42 protein results in completely processive polymerization and the generation of full-length products. Similar processivity is observed with the HSV-1 DNA polymerase purified from herpes-infected Vero cells. Processive DNA replication by the DNA polymerase isolated from HSV-1-infected Vero cells or the recombinant DNA polymerase-UL42 protein complex requires that the single-stranded DNA be coated with saturating levels of ICP8. ICP8 which binds single-stranded DNA in a highly cooperative manner is presumably required to melt out regions of secondary structure in the single-stranded DNA template, thereby potentiating the processivity enhancing action of the UL42 protein.
单纯疱疹病毒1型（HSV-1）的UL42蛋白是HSV-1基因组复制所需的七种疱疹编码多肽之一，它与HSV-1 DNA聚合酶以1:1的复合物形式存在（克鲁特，J. J.，和莱曼，I. R.（1989年）《生物化学杂志》264，19266 - 19270）。为了获得不含UL42蛋白的疱疹DNA聚合酶，我们在重组杆状病毒载体中克隆并过量表达了Pol基因，并将重组DNA聚合酶纯化至接近均一性。在疱疹编码的单链DNA结合蛋白ICP8存在的情况下，重组酶对单引物的M13mp18单链DNA进行复制，除了一些全长产物外，通过脱氧核苷酸聚合的准连续模式还产生了一系列中间长度的产物。添加纯化的UL42蛋白会导致完全连续的聚合反应并生成全长产物。从感染疱疹的Vero细胞中纯化的HSV-1 DNA聚合酶也观察到了类似的连续性。从感染HSV-1的Vero细胞中分离出的DNA聚合酶或重组DNA聚合酶 - UL42蛋白复合物进行的连续DNA复制要求单链DNA被饱和水平的ICP8覆盖。以高度协同方式结合单链DNA的ICP8可能是为了解开单链DNA模板中的二级结构区域，从而增强UL42蛋白的连续性增强作用。
