We address the role of enzyme conformational dynamics in specificity for a high-fidelity DNA polymerase responsible for genome replication. We present the complete characterization of the conformational dynamics during the correct nucleotide incorporation forward and reverse reactions using stopped-flow and rapid-quench methods with a T7 DNA polymerase variant containing a fluorescent unnatural amino acid, (7-hydroxy-4-coumarin-yl) ethylglycine, which provides a signal for enzyme conformational changes. We show that the forward conformational change (> 6000 s-1) is much faster than chemistry (300 s-1) while the enzyme opening to allow release of bound nucleotide (1.7 s-1) is much slower than chemistry. These parameters show that the conformational change selects a correct nucleotide for incorporation through an induced-fit mechanism. We also measured conformational changes occurring after chemistry and during pyrophosphorolysis, providing new insights into processive polymerization. Pyrophosphorolysis follows a conformational selection mechanism as the pyrophosphate binds to a rare pre-translocation state of the enzyme-DNA complex. Global data fitting was achieved by including experiments in the forward and reverse directions to correlate conformational changes with chemical reaction steps. This analysis provided well constrained values for nine rate constants to establish a complete free energy profile including the rates of DNA translocation during processive synthesis. Translocation does not follow Brownian ratchet or power stroke models invoking nucleotide binding as the driving force. Rather, translocation is rapid and thermodynamically favorable after enzyme opening and pyrophosphate release, and it appears to limit the rate of processive synthesis at 4° C.

中文翻译：

---

使用带有荧光人工氨基酸的 DNA 聚合酶定义的高保真 DNA 复制和易位期间的构象动力学

我们解决了酶构象动力学在负责基因组复制的高保真 DNA 聚合酶的特异性中的作用。我们使用含有荧光非天然氨基酸（7-羟基-4-香豆素基) 乙基甘氨酸，为酶构象变化提供信号。我们表明正向构象变化 (> 6000 s-1) 比化学反应 (300 s-1) 快得多，而允许释放结合核苷酸 (1.7 s-1) 的酶开放比化学反应慢得多。这些参数表明，构象变化通过诱导拟合机制选择了正确的核苷酸进行掺入。我们还测量了化学反应后和焦磷酸分解过程中发生的构象变化，为持续聚合提供了新的见解。焦磷酸分解遵循构象选择机制，因为焦磷酸盐与酶-DNA 复合物的罕见易位前状态结合。全局数据拟合是通过包括正向和反向实验以将构象变化与化学反应步骤相关联来实现的。该分析为九个速率常数提供了很好的约束值，以建立完整的自由能谱，包括持续合成过程中 DNA 易位的速率。易位不遵循以核苷酸结合为驱动力的布朗棘轮或动力冲程模型。而是，