We show that T7 DNA polymerase (pol) and exonuclease (exo) domains contribute to selective error correction during DNA replication by regulating bidirectional strand transfer between the two active sites. To explore the kinetic basis for selective removal of mismatches, we used a fluorescent cytosine analog (1,3-diaza-2-oxophenoxazine) to monitor the kinetics of DNA transfer between the exo and pol sites. We globally fit stopped-flow fluorescence and base excision kinetic data and compared results obtained with ssDNA versus duplex DNA to resolve how DNA transfer governs exo specificity. We performed parallel studies using hydrolysis-resistant phosphorothioate oligonucleotides to monitor DNA transfer to the exo site without hydrolysis. ssDNA binds to the exo site at the diffusion limit (10⁹ M⁻¹ s⁻¹, K_d = 40 nM) followed by fast hydrolysis of the 3′-terminal nucleotide (>5000 s⁻¹). Analysis using duplex DNA with a 3′-terminal mismatch or a buried mismatch exposed a unique intermediate state between pol and exo active sites and revealed that transfer via the intermediate to the exo site is stimulated by free nucleoside triphosphates. Transfer from the exo site back to the pol site after cleavage is fast and efficient. We propose a model to explain why buried mismatches are removed faster than single 3′-terminal mismatches and thereby provide an additional opportunity for error correction. Our data provide the first comprehensive model to explain how DNA transfer from pol to exo active sites and back again after base excision allow efficient selective mismatch removal during DNA replication to improve fidelity by more than 1000-fold.

我们表明，T7 DNA 聚合酶 (pol) 和核酸外切酶 (exo) 域通过调节两个活性位点之间的双向链转移，在 DNA 复制过程中有助于选择性纠错。为了探索选择性去除错配的动力学基础，我们使用荧光胞嘧啶类似物（1,3-diaza-2-oxophenoxazine）来监测 exo 和 pol 位点之间 DNA 转移的动力学。我们全局拟合停流荧光和碱基切除动力学数据，并将使用 ssDNA与双链 DNA 获得的结果进行比较，以解决 DNA 转移如何控制外切特异性。我们使用耐水解的硫代磷酸酯寡核苷酸进行了平行研究，以监测 DNA 在不水解的情况下转移到外切位点。ssDNA 在扩散极限 (10 ⁹ M ^-1 s ^-1，K _d = 40 nM)，然后快速水解 3'-末端核苷酸 (>5000 s ^-1 )。使用具有 3' 末端错配或掩埋错配的双链 DNA 进行的分析揭示了 pol 和 exo 活性位点之间的独特中间状态，并揭示了通过外切位点的中间体受到游离核苷三磷酸的刺激。切割后从 exo 站点转移回 pol 站点是快速有效的。我们提出了一个模型来解释为什么埋藏的错配比单个 3'-末端错配被移除得更快，从而提供了额外的纠错机会。我们的数据提供了第一个综合模型来解释 DNA 如何从 pol 转移到 exo 活性位点并在碱基切除后再次转移，从而在 DNA 复制过程中有效地选择性去除错配，从而将保真度提高 1000 多倍。