Huber Claudia, Marx Andreas
Department of Chemistry, University of Konstanz, Konstanz, Germany.
PLoS One. 2017 Aug 23;12(8):e0183623. doi: 10.1371/journal.pone.0183623. eCollection 2017.
Fidelity and selectivity of DNA polymerases are critical determinants for the biology of life, as well as important tools for biotechnological applications. DNA polymerases catalyze the formation of DNA strands by adding deoxynucleotides to a primer, which is complementarily bound to a template. To ensure the integrity of the genome, DNA polymerases select the correct nucleotide and further extend the nascent DNA strand. Thus, DNA polymerase fidelity is pivotal for ensuring that cells can replicate their genome with minimal error. DNA polymerases are, however, further optimized for more specific biotechnological or diagnostic applications. Here we report on the semi-rational design of mutant libraries derived by saturation mutagenesis at single sites of a 3'-5'-exonuclease deficient variant of Thermococcus kodakaraensis DNA polymerase (KOD pol) and the discovery for variants with enhanced mismatch extension selectivity by screening. Sites of potential interest for saturation mutagenesis were selected by their proximity to primer or template strands. The resulting libraries were screened via quantitative real-time PCR. We identified three variants with single amino acid exchanges-R501C, R606Q, and R606W-which exhibited increased mismatch extension selectivity. These variants were further characterized towards their potential in mismatch discrimination. Additionally, the identified enzymes were also able to differentiate between cytosine and 5-methylcytosine. Our results demonstrate the potential in characterizing and developing DNA polymerases for specific PCR based applications in DNA biotechnology and diagnostics.
DNA聚合酶的保真度和选择性是生命生物学的关键决定因素，也是生物技术应用的重要工具。DNA聚合酶通过将脱氧核苷酸添加到与模板互补结合的引物上来催化DNA链的形成。为确保基因组的完整性，DNA聚合酶会选择正确的核苷酸并进一步延伸新生的DNA链。因此，DNA聚合酶的保真度对于确保细胞能够以最小的错误复制其基因组至关重要。然而，DNA聚合酶还针对更特定的生物技术或诊断应用进行了进一步优化。在此，我们报告了通过对嗜热栖热菌DNA聚合酶（KOD pol）的3'-5'-核酸外切酶缺陷变体的单一位点进行饱和诱变而获得的突变文库的半理性设计，并通过筛选发现了具有增强的错配延伸选择性的变体。通过其与引物或模板链的接近程度来选择饱和诱变的潜在感兴趣位点。通过定量实时PCR对所得文库进行筛选。我们鉴定出了三个具有单氨基酸交换的变体——R501C、R606Q和R606W——它们表现出增加的错配延伸选择性。进一步对这些变体在错配识别方面的潜力进行了表征。此外，所鉴定的酶还能够区分胞嘧啶和5-甲基胞嘧啶。我们的结果证明了在表征和开发用于DNA生物技术和诊断中基于特定PCR应用的DNA聚合酶方面的潜力。
