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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Biochemical evidence for the requirement of Hoogsteen base pairing for replication by human DNA polymerase ι
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Biochemical evidence for the requirement of Hoogsteen base pairing for replication by human DNA polymerase ι

机译：需要Hoogsteen碱基配对才能通过人DNA聚合酶1复制的生化证据

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摘要

Because of the near geometric identity of Watson-Crick (W-C) G·C and A·T base pairs, a given DNA polymerase forms the four possible correct base pairs with nearly identical catalytic efficiencies. However, human DNA polymerase ι (Polι), a member of the Y family of DNA polymerases, exhibits a marked template specificity, being more efficient at incorporating the correct nucleotide opposite template purines than opposite pyrimidines. By using 7-deazaa-denine and 7-deazaguanine as the templating residues, which disrupt Hoogsteen base pair formation, we show that, unlike the other DNA polymerases belonging to the A, B, or Y family, DNA synthesis by Polι is severely inhibited by these N7-modified bases. These observations provide biochemical evidence that, during normal DNA synthesis, template purines adopt a syn conformation in the Polι active site, enabling the formation of a Hoogsteen base pair with the incoming pyrimidine nucleotide. Additionally, muta-tional studies with Leu-62, which lies in close proximity to the templating residue in the Polι ternary complex, have indicated that both factors, steric constraints within the active site and the stability provided by the hydrogen bonds in the Hoogsteen base pair, contribute to the efficiency of correct nucleotide incorporation opposite template purines by Polι.
机译：由于沃森-克里克（W-C）G·C和A·T碱基对的几何相似性，给定的DNA聚合酶形成了四个具有几乎相同的催化效率的正确碱基对。然而，DNA聚合酶的Y家族的成员人DNA聚合酶1（Pol 1）表现出显着的模板特异性，在掺入正确的核苷酸相反的模板嘌呤方面比相反的嘧啶更有效。通过使用7-deazaa-denine和7-deazaguan嘌呤作为模板残基，它们破坏了Hoogsteen碱基对的形成，我们证明，与属于A，B或Y家族的其他DNA聚合酶不同，Polι的DNA合成受到严重抑制这些N7修饰的碱基。这些观察提供了生物化学证据，在正常的DNA合成期间，模板嘌呤在Pol 1活性位点采用了顺式构象，从而使得能够与传入的嘧啶核苷酸形成Hoogsteen碱基对。此外，使用Leu-62进行的突变研究表明，这两个因素都非常接近Pol三元络合物中的模板残基，这两个因素包括：活性位点内的空间位阻以及Hoogsteen碱基中氢键提供的稳定性对，有助于通过PolI正确地与模板嘌呤相对地核苷酸掺入。

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