Nimonkar Amitabh V, Boehmer Paul E
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101-6129, USA.
J Biol Chem. 2004 May 21;279(21):21957-65. doi: 10.1074/jbc.M400832200. Epub 2004 Mar 16.
Recombination-dependent replication is an integral part of the process by which double-strand DNA breaks are repaired to maintain genome integrity. It also serves as a means to replicate genomic termini. We reported previously on the reconstitution of a recombination-dependent replication system using purified herpes simplex virus type 1 proteins (Nimonkar A. V., and Boehmer, P. E. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10201-10206). In this system, homologous pairing by the viral single-strand DNA-binding protein (ICP8) is coupled to DNA synthesis by the viral DNA polymerase and helicase-primase in the presence of a DNA-relaxing enzyme. Here we show that DNA synthesis in this system is dependent on the viral polymerase processivity factor (UL42). Moreover, although DNA synthesis is strictly dependent on topoisomerase I, it is only stimulated by the viral helicase in a manner that requires the helicase-loading protein (UL8). Furthermore, we have examined the dependence of DNA synthesis in the viral system on species-specific protein-protein interactions. Optimal DNA synthesis was observed with the herpes simplex virus type 1 replication proteins, ICP8, DNA polymerase (UL30/UL42), and helicase-primase (UL5/UL52/UL8). Interestingly, substitution of each component with functional homologues from other systems for the most part did not drastically impede DNA synthesis. In contrast, recombination-dependent replication promoted by the bacteriophage T7 replisome was disrupted by substitution with the replication proteins from herpes simplex virus type 1. These results show that although DNA synthesis performed by the T7 replisome is dependent on cognate protein-protein interactions, such interactions are less important in the herpes simplex virus replisome.
依赖重组的复制是双链DNA断裂修复以维持基因组完整性过程中不可或缺的一部分。它也是复制基因组末端的一种方式。我们之前报道了使用纯化的1型单纯疱疹病毒蛋白重建依赖重组的复制系统（尼蒙卡尔·A.V.和伯默尔·P.E.（2003年）《美国国家科学院院刊》100，10201 - 10206）。在这个系统中，病毒单链DNA结合蛋白（ICP8）的同源配对与病毒DNA聚合酶和螺旋酶 - 引发酶在存在DNA松弛酶的情况下的DNA合成相偶联。在这里我们表明该系统中的DNA合成依赖于病毒聚合酶持续合成因子（UL42）。此外，虽然DNA合成严格依赖于拓扑异构酶I，但它仅以需要解旋酶装载蛋白（UL8）的方式被病毒解旋酶刺激。此外，我们研究了病毒系统中DNA合成对物种特异性蛋白质 - 蛋白质相互作用的依赖性。用1型单纯疱疹病毒复制蛋白ICP8、DNA聚合酶（UL30/UL42）和螺旋酶 - 引发酶（UL5/UL52/UL8）观察到了最佳的DNA合成。有趣的是，用来自其他系统的功能同源物替换每个组分在很大程度上并没有严重阻碍DNA合成。相比之下，噬菌体T7复制体促进的依赖重组的复制被用1型单纯疱疹病毒的复制蛋白替换所破坏。这些结果表明，虽然T7复制体进行的DNA合成依赖于同源蛋白质 - 蛋白质相互作用，但这种相互作用在单纯疱疹病毒复制体中不太重要。
