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首页> 外文期刊>Journal of Molecular Biology >A bipartite polymerase-processivity factor interaction: Only the internal beta binding site of the alpha subunit is required for processive replication by the DNA polymerase III holoenzyme
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A bipartite polymerase-processivity factor interaction: Only the internal beta binding site of the alpha subunit is required for processive replication by the DNA polymerase III holoenzyme

机译：二聚体聚合酶-加工因子相互作用：DNA聚合酶III全酶进行复制仅需要alpha亚基的内部β结合位点

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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.
机译：以前，我们将DNA聚合酶III的催化亚基（alpha）的beta（2）相互作用部分定位到聚合酶活性位点下游的C端一半。从那时起，已经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），并且极端的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，我们在两个位点都进行了突变，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，这是在非进行性填补缺口的测定中进行的。内部位点的突变消除了突变体α亚基参与过程合成的能力。用AAAKK取代五残基内部序列消除了与beta（2）的可检测结合。另外，β（2）结合所需的残基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-末端位点的突变表现出接近野生型的表型。具有完全去除C末端位点的alpha亚基可以参与过程性DNA复制，可以结合beta（2），并且如果被诱导为高水平表达，则可以补充对温度敏感的条件致死性dnaE突变。仅部分互补的C端缺陷与结合到tau而不是beta（2）的缺陷相关。 C端删除只减少beta（2）绑定四倍; tau结合减少了约400倍。出现了beta（2）结合位点的上下文产生了巨大的差异。用共有的beta（2）结合序列替换内部位点可将生成的alpha对beta 2的亲和力提高100倍以上，而在C端位点进行相同的修饰不会显着增加结合。讨论了复制酶及其持续性因子之间多重相互作用的含义，包括应用于聚合酶循环以及在复制叉处与其他聚合酶和因子互换。 （c）2005 Elsevier Ltd.保留所有权利。

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