Metal-dependent conformational activation explains highly promutagenic replication across O6-methylguanine by human DNA polymerase β.
Metal-dependent conformational activation explains highly promutagenic replication across O6-methylguanine by human DNA polymerase β.
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DOI：
10.1021/ja500172d
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发表时间：
2014-04-16
影响因子：
15
通讯作者：
Lee S
中科院分区：
文献类型：
--
作者：
Koag MC;Lee S
人类DNA聚合酶β (polβ)插入致癌病变o6 -甲基鸟嘌呤（O6MeG）的频率是c的30倍，尽管速度较慢。为了深入了解这一促生过程，我们在活性位点Mg2+或Mn2+存在的情况下，用dCTP或dTTP类似物碱基与O6MeG配对，解决了polβ的四个三元结构。Mg2+结合结构表明，O6MeG·dCTP/ dTTP-Mg2 +配合物均采用开放的蛋白质构象、交错的碱基对和一个活性位点的金属离子。Mn2+结合结构表明，O6Me·dCTP-Mn2 +复合物具有类似的改变构象，而O6MeG·dTTP-Mn2 +复合物具有封闭的蛋白质构象和伪沃森-克里克碱基对，具有催化活性态。在这些观察的基础上，我们得出结论，pol - β通过诱导非最佳催化构象的改变来减缓与O6MeG相反的核苷酸整合，并通过在酶活性位点允许O6MeG·T的沃森-克里克模式而不是O6MeG·C的沃森-克里克模式促进诱变复制。O6MeG·dTTP-Mn2 +三元结构，代表了第一个具有封闭蛋白质构象和共面碱基对的错配的polβ三元配合物结构，第一个在DNA聚合酶活性位点形成的伪沃森-克里克O6MeG·T结构，以及罕见的，如果不是第一个，金属依赖的DNA聚合酶构象激活的例子。表明催化金属离子配位是polβ的动力学检查点，对polβ的构象活化至关重要。总的来说，我们的结构研究不仅解释了polβ在O6MeG中的促生催化作用，而且为polβ的复制保真度提供了新的见解。
Human DNA polymerase β (polβ) inserts, albeit slowly, T opposite the carcinogenic lesion O6-methylguanine (O6MeG) ∼30-fold more frequently than C. To gain insight into this promutagenic process, we solved four ternary structures of polβ with an incoming dCTP or dTTP analogue base-paired with O6MeG in the presence of active-site Mg2+ or Mn2+. The Mg2+-bound structures show that both the O6MeG·dCTP/dTTP–Mg2+ complexes adopt an open protein conformation, staggered base pair, and one active-site metal ion. The Mn2+-bound structures reveal that, whereas the O6Me·dCTP–Mn2+ complex assumes the similar altered conformation, the O6MeG·dTTP–Mn2+ complex adopts a catalytically competent state with a closed protein conformation and pseudo-Watson–Crick base pair. On the basis of these observations, we conclude that polβ slows nucleotide incorporation opposite O6MeG by inducing an altered conformation suboptimal for catalysis and promotes mutagenic replication by allowing Watson–Crick-mode for O6MeG·T but not for O6MeG·C in the enzyme active site. The O6MeG·dTTP–Mn2+ ternary structure, which represents the first structure of mismatched polβ ternary complex with a closed protein conformation and coplanar base pair, the first structure of pseudo-Watson–Crick O6MeG·T formed in the active site of a DNA polymerase, and a rare, if not the first, example of metal-dependent conformational activation of a DNA polymerase, indicate that catalytic metal-ion coordination is utilized as a kinetic checkpoint by polβ and is crucial for the conformational activation of polβ. Overall, our structural studies not only explain the promutagenic polβ catalysis across O6MeG but also provide new insights into the replication fidelity of polβ.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
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