A bipartite polymerase-processivity factor interaction:: Only the internal β binding site of the α subunit is required for processive replication by the DNA polymerase III holoenzyme
A bipartite polymerase-processivity factor interaction:: Only the internal β binding site of the α subunit is required for processive replication by the DNA polymerase III holoenzyme
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DOI：
10.1016/j.jmb.2005.04.065
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发表时间：
2005-07-08
影响因子：
5.6
通讯作者：
McHenry, CS
中科院分区：
文献类型：
--
作者：
Dohrmann, PR;McHenry, CS
此前，我们将 DNA 聚合酶 III 催化亚基 (α) 的 β(2) 相互作用部分定位于聚合酶活性位点下游的 C 端一半。从那时起，人们提出了该区域内两个不同的 beta(2) 结合位点。内部位点包括氨基酸残基920-924 (QADMF)，并且极端C末端位点包括氨基酸残基1154-1159(.)(QVELEF)。为了确定它们的相对贡献，我们在两个位点进行了突变，并评估了突变体 a 亚基的生化、遗传和蛋白质结合特性。所有纯化的突变体α亚基都保留了接近野生型聚合酶的功能，这是在非进行性间隙填充测定中测量的。内部位点的突变消除了突变体α亚基参与持续合成的能力。用 AAAKK 替换五残基内部序列消除了可检测到的与 beta(2) 的结合。此外，β(2)结合所需残基的突变消除了所得聚合酶参与体内染色体复制的能力。相反，C 端位点的突变表现出接近野生型的表型。 C 端位点完全去除的 α 亚基可以参与持续的 DNA 复制，可以结合 beta(2)，并且如果诱导高水平表达，可以补充温度敏感的条件致死 dnaE 突变。仅部分互补的 C 端缺陷与 tau 结合缺陷相关，而不是与 beta(2) 结合缺陷相关。 C 端删除仅使 beta(2) 结合减少四倍； tau 结合减少约 400 倍。 beta(2) 结合位点的出现环境产生了巨大的差异。用共有的 β(2) 结合序列替换内部位点，所得 α 对 β 2 的亲和力增加了 100 倍以上，而 C 端位点的相同修饰并没有显着增加结合。讨论了复制酶与其持续因子之间的多重相互作用的影响，包括在聚合酶循环中的应用以及在复制叉处与其他聚合酶和因子的互换。 (c) 2005 Elsevier Ltd. 保留所有权利。
Previously, we localized the beta(2) interacting portion of the catalytic subunit (alpha) of DNA polymerase III to the C-terminal half, downstream of the polymerase active site. Since then, two different beta(2) binding sites within this region have been proposed. An internal site includes amino acid residues 920-924 (QADMF) and an extreme C-terminal site includes amino acid residues 1154-1159 (.)(QVELEF). To permit determination of their relative contributions, we made mutations in both sites and evaluated the biochemical, genetic, and protein binding properties of the mutant a subunits. All purified mutant alpha subunits retained near wild-type polymerase function, which was measured in non-processive gap-filling assays. Mutations in the internal site abolished the ability of mutant alpha subunits to participate in processive synthesis. Replacement of the five-residue internal sequence with AAAKK eliminated detectable binding to beta(2). In addition, mutation of residues required for beta(2) binding abolished the ability of the resulting polymerase to participate in chromosomal replication in vivo. In contrast, mutations in the C-terminal site exhibited near wild-type phenotypes. alpha Subunits with the C-terminal site completely removed could participate in processive DNA replication, could bind beta(2), and, if induced to high level expression, could complement a temperaturesensitive conditional lethal dnaE mutation. C-terminal defects that only partially complemented correlated with a defect in binding to tau, not beta(2). A C-terminal deletion only reduced beta(2) binding fourfold; tau binding was decreased ca 400-fold. The context in which the beta(2) binding site was presented made an enormous difference. Replacement of the internal site with a consensus beta(2) binding sequence increased the affinity of the resulting alpha for beta 2 over 100-fold, whereas the same modification at the C-terminal site did not significantly increase binding. The implications of multiple interactions between a replicase and its processivity factor, including applications to polymerase cycling and interchange with other polymerases and factors at the replication fork, are discussed. (c) 2005 Elsevier Ltd. All rights reserved.