Horhota Allen, Zou Keyong, Ichida Justin K, Yu Biao, McLaughlin Larry W, Szostak Jack W, Chaput John C
Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02467, USA.
J Am Chem Soc. 2005 May 25;127(20):7427-34. doi: 10.1021/ja0428255.
alpha-l-Threofuranosyl nucleoside triphosphates (tNTPs) are tetrafuranose nucleoside derivatives and potential progenitors of present-day beta-d-2'-deoxyribofuranosyl nucleoside triphosphates (dNTPs). Therminator DNA polymerase, a variant of the 9 degrees N DNA polymerase, is an efficient DNA-directed threosyl nucleic acid (TNA) polymerase. Here we report a detailed kinetic comparison of Therminator-catalyzed TNA and DNA syntheses. We examined the rate of single-nucleotide incorporation for all four tNTPs and dNTPs from a DNA primer-template complex and carried out parallel experiments with a chimeric DNA-TNA primer-DNA template containing five TNA residues at the primer 3'-terminus. Remarkably, no drop in the rate of TNA incorporation was observed in comparing the DNA-TNA primer to the all-DNA primer, suggesting that few primer-enzyme contacts are lost with a TNA primer. Moreover, comparison of the catalytic efficiency of TNA synthesis relative to DNA synthesis at the downstream positions reveals a difference of no greater than 5-fold in favor of the natural DNA substrate. This disparity becomes negligible when the TNA synthesis reaction mixture is supplemented with 1.25 mM MnCl(2). These results indicate that Therminator DNA polymerase can recognize both a TNA primer and tNTP substrates and is an effective catalyst of TNA polymerization despite changes in the geometry of the reactants.
α-L-苏糖呋喃糖核苷三磷酸（tNTPs）是四呋喃糖核苷衍生物，也是当今β-D-2'-脱氧核糖呋喃糖核苷三磷酸（dNTPs）的潜在前体。嗜热栖热菌DNA聚合酶是9°N DNA聚合酶的变体，是一种高效的DNA定向苏糖核酸（TNA）聚合酶。在此，我们报告了嗜热栖热菌催化的TNA和DNA合成的详细动力学比较。我们检测了DNA引物-模板复合物中所有四种tNTPs和dNTPs的单核苷酸掺入速率，并对在引物3'-末端含有五个TNA残基的嵌合DNA-TNA引物-DNA模板进行了平行实验。值得注意的是，将DNA-TNA引物与全DNA引物进行比较时，未观察到TNA掺入速率下降，这表明TNA引物几乎没有失去引物-酶接触。此外，比较下游位置TNA合成相对于DNA合成的催化效率，发现有利于天然DNA底物的差异不超过5倍。当TNA合成反应混合物补充1.25 mM MnCl₂时，这种差异可以忽略不计。这些结果表明，嗜热栖热菌DNA聚合酶既能识别TNA引物，也能识别tNTP底物，尽管反应物的几何形状发生了变化，但它仍是TNA聚合的有效催化剂。
