BACKGROUND The discovery of thermostable DNA polymerases such as Taq DNA polymerase revolutionized amplification of DNA by polymerase chain reaction methods that rely on thermal cycling for strand separation. These methods are widely used in the laboratory for medical research, clinical diagnostics, criminal forensics and general molecular biology research. Today there is a growing demand for on-site molecular diagnostics; so-called 'Point-of-Care tests'. Isothermal nucleic acid amplification techniques do not require a thermal cycler making these techniques more suitable for performing Point-of-Care tests at ambient temperatures compared to traditional polymerase chain reaction methods. Strand-displacement activity is essential for such isothermal nucleic acid amplification; however, the selection of DNA polymerases with inherent strand-displacement activity that are capable of performing DNA synthesis at ambient temperatures is currently limited. RESULTS We have characterized the large fragment of a DNA polymerase I originating from the marine psychrophilic bacterium Psychrobacillus sp. The enzyme showed optimal polymerase activity at pH 8-9 and 25-110 mM NaCl/KCl. The polymerase was capable of performing polymerase as well as robust strand-displacement DNA synthesis at ambient temperatures (25-37 °C). Through molecular evolution and screening of thousand variants we have identified a single amino-acid exchange of Asp to Ala at position 422 which induced a 2.5-fold increase in strand-displacement activity of the enzyme. Transferring the mutation of the conserved Asp residue to corresponding thermophilic homologues from Ureibacillus thermosphaericus and Geobacillus stearothermophilus also resulted in a significant increase in the strand-displacement activity of the enzymes. CONCLUSIONS Substituting Asp with Ala at positon 422 resulted in a significant increase in strand-displacement activity of three prokaryotic A-family DNA polymerases adapted to different environmental temperatures i.e. being psychrophilic and thermophilic of origin. This strongly indicates an important role for the 422 position and the O1-helix for strand-displacement activity of DNA polymerase I. The D422A variants generated here may be highly useful for isothermal nucleic acid amplification at a wide temperature scale.

中文翻译：

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DNA聚合酶的表征和工程设计揭示了手指亚结构域中的单个氨基酸取代，从而增加了A系列原核DNA聚合酶的链置换活性。

背景技术诸如Taq DNA聚合酶的热稳定DNA聚合酶的发现通过依赖于热循环进行链分离的聚合酶链反应方法彻底改变了DNA的扩增。这些方法广泛用于实验室的医学研究，临床诊断，刑事取证和一般分子生物学研究。如今，对现场分子诊断的需求不断增长。所谓的“现场护理测试”。等温核酸扩增技术不需要热循环仪，因此与传统的聚合酶链反应方法相比，这些技术更适合在环境温度下进行即时检测。链置换活性对于这种等温核酸扩增是必不可少的。然而，目前，在环境温度下能够进行DNA合成的具有固有链置换活性的DNA聚合酶的选择受到限制。结果我们表征了源自海洋嗜冷细菌Psychrobacillus sp。的DNA聚合酶I的大片段。该酶在pH 8-9和25-110 mM NaCl / KCl下显示最佳聚合酶活性。该聚合酶能够在环境温度（25-37°C）下执行聚合酶以及强大的链置换DNA合成。通过分子进化和数千种变体的筛选，我们确定了Asp在422位氨基酸向Ala的单个氨基酸交换，这导致该酶的链置换活性增加了2.5倍。将保守的Asp残基的突变转移至来自嗜热球菌和嗜热脂肪地芽孢杆菌的相应嗜热同源物，也导致酶的链置换活性显着增加。结论在422位正电子上用Ala取代Asp导致三种原核A族DNA聚合酶的链置换活性显着增加，这些酶适应于不同的环境温度，即起源于亲热和嗜热。这强烈表明422位置和O1-螺旋对于DNA聚合酶I的链置换活性具有重要作用。此处产生的D422A变体对于在宽温度范围内等温核酸扩增可能非常有用。