Mutations to create thermostable reverse transcriptase with bacterial family A DNA polymerase from Thermotoga petrophila K4
Mutations to create thermostable reverse transcriptase with bacterial family A DNA polymerase from Thermotoga petrophila K4
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DOI：
10.1016/j.jbiosc.2011.11.001
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发表时间：
2012-03-01
影响因子：
2.8
通讯作者：
Fujiwara, Shinsuke
中科院分区：
文献类型：
--
作者：
Sano, Sotaro;Yamada, Yosuke;Fujiwara, Shinsuke
从嗜岩栖热袍菌（Thermotoga petrophila）K4中获得了重组A家族DNA聚合酶（K4 Poll），并对其酶学性质进行了分析。K4 Poll显示具有3 '-5'核酸外切酶活性的热稳定的DNA依赖性DNA聚合酶活性，但没有可检测到的RNA依赖性DNA聚合酶活性。通过计算机模拟推测其三级结构，以了解K4 Poll与模板DNA之间的结合情况。预测3 '-5'核酸外切酶结构域中的9个氨基酸通过与核糖的2'羟基的空间干扰参与DNA/RNA区分。为了允许K4 Poll接受RNA作为模板，构建了突变体，重点是位于结合核糖的2'羟基周围的氨基酸。其中Thr 326、Leu 329、Gln 384、Phe 388、Met 408或Tyr 438被Ala替换的突变体（分别命名为T326 A、L329 A、Q384 A、F388 A、M408 A或Y 438 A）显示RNA依赖性DNA聚合酶活性。所有突变体显示出降低的3 '-5'核酸外切酶活性，表明逆转录酶活性的获得与3 '-5'核酸外切酶活性的丧失相关。特别地，突变体能够在单管形式中从结构化RNA直接扩增DNA，所述结构化RNA不能被逆转录病毒逆转录酶有效扩增。(C)2011年，生物技术学会，日本。All rights reserved.
Family A DNA polymerase (K4Poll) from Thermotoga petrophila K4 was obtained as a recombinant form, and the enzyme characteristics were analyzed. K4Poll showed thermostable DNA-dependent DNA polymerase activity with 3'-5' exonuclease activity but no detectable RNA-dependent DNA polymerase activity. Its tertiary structure was speculated by in silico modeling to understand the binding situation between K4Poll and template DNA. Nine amino acids in the 3'-5' exonuclease domain are predicted to be involved in DNA/RNA distinction by steric interference with the 2' hydroxy group of ribose. To allow K4Poll to accept RNA as the template, mutants were constructed focusing on the amino acids located around the 2' hydroxyl group of the bound ribose. The mutants in which Thr326, Leu329, Gln384, Phe388, Met408, or Tyr438 was replaced with Ala (designated as T326A, L329A, Q384A, F388A, M408A, or Y438A, respectively) showed RNA-dependent DNA polymerase activity. All the mutants showed reduced 3'-5' exonuclease activity, suggesting that gain of reverse transcriptase activity is correlated with loss of 3'-5' exonuclease activity. In particular, the mutants enabled direct DNA amplification in a single tube format from structured RNA that was not efficiently amplified by retroviral reverse transcriptase. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.