Department of Molecular Microbial and Structural Biology, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06085, USA.
J Virol. 2011 Jan;85(2):968-78. doi: 10.1128/JVI.01690-10. Epub 2010 Nov 10.
The heterotrimeric helicase-primase complex of herpes simplex virus type I (HSV-1), consisting of UL5, UL8, and UL52, possesses 5' to 3' helicase, single-stranded DNA (ssDNA)-dependent ATPase, primase, and DNA binding activities. In this study we confirm that the UL5-UL8-UL52 complex has higher affinity for forked DNA than for ssDNA and fails to bind to fully annealed double-stranded DNA substrates. In addition, we show that a single-stranded overhang of greater than 6 nucleotides is required for efficient enzyme loading and unwinding. Electrophoretic mobility shift assays and surface plasmon resonance analysis provide additional quantitative information about how the UL5-UL8-UL52 complex associates with the replication fork. Although it has previously been reported that in the absence of DNA and nucleoside triphosphates the UL5-UL8-UL52 complex exists as a monomer in solution, we now present evidence that in the presence of forked DNA and AMP-PNP, higher-order complexes can form. Electrophoretic mobility shift assays reveal two discrete complexes with different mobilities only when helicase-primase is bound to DNA containing a single-stranded region, and surface plasmon resonance analysis confirms larger amounts of the complex bound to forked substrates than to single-overhang substrates. Furthermore, we show that primase activity exhibits a cooperative dependence on protein concentration while ATPase and helicase activities do not. Taken together, these data suggest that the primase activity of the helicase-primase requires formation of a dimer or higher-order structure while ATPase activity does not. Importantly, this provides a simple mechanism for generating a two-polymerase replisome at the replication fork.
单纯疱疹病毒 I 型（HSV-1）的异源三聚体解旋酶-引发酶复合物由 UL5、UL8 和 UL52 组成，具有 5' 到 3' 解旋酶、单链 DNA（ssDNA）依赖性 ATP 酶、引发酶和 DNA 结合活性。在这项研究中，我们证实 UL5-UL8-UL52 复合物对分叉 DNA 的亲和力高于 ssDNA，并且无法结合完全退火的双链 DNA 底物。此外，我们表明，需要大于 6 个核苷酸的单链突出才能有效加载和展开酶。电泳迁移率变动分析和表面等离子体共振分析提供了有关 UL5-UL8-UL52 复合物与复制叉结合的更多定量信息。尽管先前有报道称在没有 DNA 和核苷三磷酸的情况下，UL5-UL8-UL52 复合物以单体形式存在于溶液中，但我们现在提供的证据表明，在存在分叉 DNA 和 AMP-PNP 的情况下，可形成更高阶的复合物。电泳迁移率变动分析仅在解旋酶-引发酶与含有单链区域的 DNA 结合时揭示出两种具有不同迁移率的离散复合物，而表面等离子体共振分析证实，与单突出底物相比，更多的复合物结合到分叉底物上。此外，我们表明，引发酶活性表现出对蛋白质浓度的协同依赖性，而 ATP 酶和解旋酶活性则没有。总之，这些数据表明，解旋酶-引发酶的引发酶活性需要形成二聚体或更高阶结构，而 ATP 酶活性则不需要。重要的是，这为在复制叉处生成双聚合酶复制体提供了一个简单的机制。
