Tenney D J, Hurlburt W W, Micheletti P A, Bifano M, Hamatake R K
Department of Virology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000.
J Biol Chem. 1994 Feb 18;269(7):5030-5.
The herpes simplex virus type 1 (HSV) UL5, UL8, and UL52 proteins form a helicase-primase complex in infected cells. Several laboratories have demonstrated that helicase and nucleoside triphosphatase activities of the heterotrimer (UL5/8/52) are indistinguishable from that of a subassembly of UL5 and UL52 (UL5/52). Although the UL5/52 subassembly functions in coupled primase-polymerase assays on homopolymeric templates, its activity on natural DNA templates has been reported to require UL8. To determine the role of UL8 in primase assays, the activity of the UL5/52 subassembly was compared to that of the heterotrimer reconstituted by adding UL8 to UL5/52. We detected significant activity of the UL5/52 subassembly in coupled primase-polymerase and oligoribonucleotide primer synthesis assays on phi X174 and M13 virion DNAs. However the addition of UL8 to UL5/52 stimulated this activity in a dose-dependent manner. We demonstrate that stimulation occurred at the level of primer synthesis. UL8 did not affect the amount or size of primers annealed to template, their utilization by DNA polymerase, or the use of specific initiation sites within the template. In kinetic studies, the rate of primer synthesis was increased by UL8 but the Km for phi X174 DNA template was unchanged. These results suggest that a function of the UL8 component of the HSV helicase-primase complex is to increase the efficiency of primer synthesis by UL5/52.
单纯疱疹病毒1型（HSV）的UL5、UL8和UL52蛋白在受感染细胞中形成解旋酶-引发酶复合物。几个实验室已证明，异源三聚体（UL5/8/52）的解旋酶和核苷三磷酸酶活性与UL5和UL52的一个亚组件（UL5/52）的活性无法区分。尽管UL5/52亚组件在同聚模板的偶联引发酶-聚合酶测定中起作用，但据报道其在天然DNA模板上的活性需要UL8。为了确定UL8在引发酶测定中的作用，将UL5/52亚组件的活性与通过向UL5/52中添加UL8重构的异源三聚体的活性进行了比较。我们在针对φX174和M13病毒粒子DNA的偶联引发酶-聚合酶和寡核糖核苷酸引物合成测定中检测到了UL5/52亚组件的显著活性。然而，向UL5/52中添加UL8以剂量依赖的方式刺激了这种活性。我们证明这种刺激发生在引物合成水平。UL8不影响与模板退火的引物的数量或大小、DNA聚合酶对它们的利用，或模板内特定起始位点的使用。在动力学研究中，UL8提高了引物合成速率，但φX174 DNA模板的米氏常数不变。这些结果表明，HSV解旋酶-引发酶复合物的UL8组分的一个功能是提高UL5/52引物合成的效率。
