Leading and lagging strand DNA synthesis in vitro by a reconstituted herpes simplex virus type 1 replisome

The synthesis of double-stranded DNA by a rolling circle mechanism was reconstituted in vitro with a replisome consisting of the DNA polymerase-UL42 complex and the heterotrimeric helicase-primase encoded by herpes simplex virus type 1. Okazaki fragments 3 kilobases in length and leading strands that may exceed 10 kilobases are produced. Lagging strand synthesis is stimulated by ribonucleoside triphosphates. DNA replication appears to be processive because it resists competition with an excess of (dT)(150)/(dA)(20). The single-strand DNA binding protein ICP8 is not required, and high concentrations of ICP8 can, in fact, inhibit lagging strand synthesis. The inhibition can, however, be overcome by the addition of an excess of the UL8 component of the helicase-primase. Rolling circle replication by the herpesvirus and bacteriophage T7 replisomes appears to proceed by a similar mechanism.

滚环复制机制介导的双链DNA合成，已通过由DNA聚合酶-UL42复合物以及1型单纯疱疹病毒编码的异三聚体解旋酶-引物酶（helicase-primase）组成的复制体（replisome）在体外成功重构。实验可生成长度为3千碱基的冈崎片段，以及长度可超过10千碱基的前导链。核糖核苷三磷酸可促进滞后链的合成。该DNA复制过程呈现持续合成特性，因其可耐受过量(dT)₁₅₀/(dA)₂₀的竞争抑制。单链DNA结合蛋白ICP8（single-strand DNA binding protein ICP8）并非必需组分，实际上高浓度的ICP8反而会抑制滞后链的合成。不过，通过添加过量的解旋酶-引物酶UL8亚基，即可克服该抑制效应。疱疹病毒与噬菌体T7复制体所介导的滚环复制，其运作机制似乎与此高度相似。