Dual functions of single-stranded DNA-binding protein in helicase loading at the bacteriophage T4 DNA replication fork
Dual functions of single-stranded DNA-binding protein in helicase loading at the bacteriophage T4 DNA replication fork
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DOI：
10.1074/jbc.m311738200
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发表时间：
2004-04-30
影响因子：
4.8
通讯作者：
Morrical, SW
中科院分区：
文献类型：
--
作者：
Ma, YJ;Wang, TS;Morrical, SW
由噬菌体 T4 DNA 聚合酶全酶催化的半保守 DNA 合成反应由链置换机制引发，需要 gp32（T4 单链 DNA (ssDNA) 结合蛋白）来隔离置换的链。启动后，新生复制叉获得 DNA 解旋酶，导致前导链 DNA 合成的速率和持续合成能力显着增加。体外研究已证实，两种 T4 编码的 DNA 解旋酶（gp41 或 dda）中的任何一种都能够刺激链置换合成。新生复制叉对任一解旋酶的获取均受到复制叉的其他蛋白质成分的调节，包括 gp32，对于 gp41 解旋酶，还受其介体/负载蛋白 gp59 的调节。在这里，我们使用蛋白质-蛋白质或蛋白质-ssDNA 相互作用缺陷的 gp32 的改变形式，分别检查 T4 复制过程中 gp32 与 gp41/gp59 和 dda 解旋酶系统之间的关系。我们表明，gp41/gp59 解旋酶对 DNA 合成的最佳刺激需要 gp32-gp59 相互作用，并且强烈依赖于 gp32 结合 ssDNA 的稳定性。荧光分析表明 gp59 按化学计量与分叉 DNA 分子结合；然而，gp59 叉状 DNA 复合物通过与 gp32 C 端“A 结构域”片段的蛋白质-蛋白质相互作用而不稳定。这些和之前发表的结果表明，在一个模型中，与滞后链 ssDNA 结合的移动 gp59-gp32 簇是 gp41 解旋酶组装的目标。相比之下，dda 解旋酶刺激 DNA 合成需要直接的 gp32-dda 蛋白质-蛋白质相互作用，并且相对不受 gp32 突变的影响，而 gp32 突变会破坏其 ssDNA 结合活性。后一数据支持这样一种模型，其中蛋白质-蛋白质与 gp32 的相互作用将 dda 维持在适当的活性状态，以便在复制叉处易位。 T4 复制中 dda 和 gp32 蛋白之间的关系似乎与疱疹病毒复制中 UL9 解旋酶和 ICP8 ssDNA 结合蛋白之间观察到的关系相似。
Semi-conservative DNA synthesis reactions catalyzed by the bacteriophage T4 DNA polymerase holoenzyme are initiated by a strand displacement mechanism requiring gp32, the T4 single-stranded DNA (ssDNA)-binding protein, to sequester the displaced strand. After initiation, DNA helicase acquisition by the nascent replication fork leads to a dramatic increase in the rate and processivity of leading strand DNA synthesis. In vitro studies have established that either of two T4-encoded DNA helicases, gp41 or dda, is capable of stimulating strand displacement synthesis. The acquisition of either helicase by the nascent replication fork is modulated by other protein components of the fork including gp32 and, in the case of the gp41 helicase, its mediator/ loading protein gp59. Here, we examine the relationships between gp32 and the gp41/gp59 and dda helicase systems, respectively, during T4 replication using altered forms of gp32 defective in either protein-protein or protein-ssDNA interactions. We show that optimal stimulation of DNA synthesis by gp41/gp59 helicase requires gp32-gp59 interactions and is strongly dependent on the stability of ssDNA binding by gp32. Fluorescence assays demonstrate that gp59 binds stoichiometrically to forked DNA molecules; however, gp59-forked DNA complexes are destabilized via protein-protein interactions with the C-terminal "A-domain" fragment of gp32. These and previously published results suggest a model in which a mobile gp59-gp32 cluster bound to lagging strand ssDNA is the target for gp41 helicase assembly. In contrast, stimulation of DNA synthesis by dda helicase requires direct gp32-dda protein-protein interactions and is relatively unaffected by mutations in gp32 that destabilize its ssDNA binding activity. The latter data support a model in which protein-protein interactions with gp32 maintain dda in a proper active state for translocation at the replication fork. The relationship between dda and gp32 proteins in T4 replication appears similar to the relationship observed between the UL9 helicase and ICP8 ssDNA-binding protein in herpesvirus replication.