A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro
A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro
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DOI：
10.1093/nar/gkh271
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发表时间：
2004-02-01
影响因子：
14.9
通讯作者：
Vander Horn, PB
中科院分区：
文献类型：
--
作者：
Wang, Y;Prosen, DE;Vander Horn, PB
允许复制 DNA 聚合酶获得高持续合成能力的机制通常是特定聚合酶特有的，不能推广到其他聚合酶。在此，我们报告了一种基于蛋白质工程的方法，通过将聚合酶结构域与序列非特异性 dsDNA 结合蛋白共价连接，显着提高 DNA 聚合酶的持续合成能力。使用来自硫磺硫化叶菌的 Sso7d 作为 DNA 结合蛋白，我们证明 A 家族和 B 家族聚合酶的持续合成能力都可以显着增强。通过在 Sso7d 中引入点突变，我们表明 Sso7d 的 dsDNA 结合特性对于增强效果至关重要。我们提出了支持两个新颖结论的证据。首先，异源 dsDNA 结合蛋白与聚合酶的融合可以提高持续合成能力，而不会影响催化活性和酶稳定性。其次，聚合酶的持续合成能力限制了 PCR 的效率，因此融合酶在 PCR 应用中比未修饰的酶表现出显着的优势。该技术有可能广泛提高核酸修饰酶的性能。
Mechanisms that allow replicative DNA polymerases to attain high processivity are often specific to a given polymerase and cannot be generalized to others. Here we report a protein engineering-based approach to significantly improve the processivity of DNA polymerases by covalently linking the polymerase domain to a sequence non-specific dsDNA binding protein. Using Sso7d from Sulfolobus solfataricus as the DNA binding protein, we demonstrate that the processivity of both family A and family B polymerases can be significantly enhanced. By introducing point mutations in Sso7d, we show that the dsDNA binding property of Sso7d is essential for the enhancement. We present evidence supporting two novel conclusions. First, the fusion of a heterologous dsDNA binding protein to a polymerase can increase processivity without compromising catalytic activity and enzyme stability. Second, polymerase processivity is limiting for the efficiency of PCR, such that the fusion enzymes exhibit profound advantages over unmodified enzymes in PCR applications. This technology has the potential to broadly improve the performance of nucleic acid modifying enzymes.