Coordinated leading and lagging strand DNA synthesis by using the herpes simplex virus 1 replication complex and minicircle DNA templates.
Coordinated leading and lagging strand DNA synthesis by using the herpes simplex virus 1 replication complex and minicircle DNA templates.
复制标题
使用单纯疱疹病毒 1 复制复合物和小环 DNA 模板协调前导链和滞后链 DNA 合成。
DOI：
10.1128/jvi.01688-10
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发表时间：
2011
影响因子：
5.4
通讯作者：
Kuchta,RobertD
中科院分区：
文献类型：
--
作者：
Stengel,Gudrun;Kuchta,RobertD
单纯疱疹病毒1型基因组的起源特异性复制需要七种蛋白质：解旋酶-引发酶（UL 5-UL 8-UL 52）、DNA聚合酶（UL 30-UL 42）、单链DNA结合蛋白（ICP 8）和起源结合蛋白（UL 9）。我们重建这些蛋白质，不包括UL 9，在合成的微环DNA模板和监测领先和滞后链DNA合成使用链特异性掺入dTMP和dAMP。导致有效的前导和滞后链合成的测定的关键特征包括高解旋酶-引发酶浓度和其序列类似于病毒DNA的滞后链模板。根据小环模板的性质，复制复合物以1：1至3：1的摩尔比合成前导链和滞后链产物。滞后链产物（0.2至0.6 kb）明显短于前导链产物（0.2至10 kb），刺激引物合成的条件导致较短的滞后链产物。ICP 8不是必需的;然而，它的存在刺激DNA合成，并增加前导链和滞后链产物的长度。奇怪的是，人类DNA聚合酶α（p70-p180或p49-p58-p70-p180），提高了疱疹病毒引物酶在线性DNA模板上合成的RNA引物的利用率，对小环的复制没有影响。缺乏聚合酶α的刺激表明存在一种大分子组装体，该组装体增强了RNA引物的利用，并可能在功能上偶联前导链和滞后链的合成。我们的观察结果进一步提供了功能偶联的证据，即（i）前导链和滞后链合成产生等量的DNA，（ii）前导链合成在滞后链上禁用引物合成的条件下进行得更快，以及（iii）加速解旋酶催化的DNA解旋的条件刺激解偶联的前导链合成，但不协调前导链和滞后链合成。
The origin-specific replication of the herpes simplex virus 1 genome requires seven proteins: the helicase-primase (UL5-UL8-UL52), the DNA polymerase (UL30-UL42), the single-strand DNA binding protein (ICP8), and the origin-binding protein (UL9). We reconstituted these proteins, excluding UL9, on synthetic minicircular DNA templates and monitored leading and lagging strand DNA synthesis using the strand-specific incorporation of dTMP and dAMP. Critical features of the assays that led to efficient leading and lagging stand synthesis included high helicase-primase concentrations and a lagging strand template whose sequence resembled that of the viral DNA. Depending on the nature of the minicircle template, the replication complex synthesized leading and lagging strand products at molar ratios varying between 1:1 and 3:1. Lagging strand products (∼0.2 to 0.6 kb) were significantly shorter than leading strand products (∼2 to 10 kb), and conditions that stimulated primer synthesis led to shorter lagging strand products. ICP8 was not essential; however, its presence stimulated DNA synthesis and increased the length of both leading and lagging strand products. Curiously, human DNA polymerase α (p70-p180 or p49-p58-p70-p180), which improves the utilization of RNA primers synthesized by herpesvirus primase on linear DNA templates, had no effect on the replication of the minicircles. The lack of stimulation by polymerase α suggests the existence of a macromolecular assembly that enhances the utilization of RNA primers and may functionally couple leading and lagging strand synthesis. Evidence for functional coupling is further provided by our observations that (i) leading and lagging strand synthesis produce equal amounts of DNA, (ii) leading strand synthesis proceeds faster under conditions that disable primer synthesis on the lagging strand, and (iii) conditions that accelerate helicase-catalyzed DNA unwinding stimulate decoupled leading strand synthesis but not coordinated leading and lagging strand synthesis.