Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Street 32, Moscow, Russia, 119991.
Mol Biotechnol. 2018 Dec;60(12):879-886. doi: 10.1007/s12033-018-0124-1.
The transferase activity of non-proofreading DNA polymerases is a well-known phenomenon that has been utilized in cloning and sequencing applications. The non-templated addition of modified nucleotides at DNA blunt ends is a potentially useful feature of DNA polymerases that can be used for selective transformation of DNA 3' ends. In this paper, we characterized the tailing reaction at perfectly matched and mismatched duplex ends with Cy3- and Cy5-modified pyrimidine nucleotides. It was shown that the best DNA tailing substrate does not have a perfect Watson-Crick base pair at the end. Mismatched duplexes with a 3' dC were the most efficient in the Taq DNA polymerase-catalysed tailing reaction with a Cy5-modified dUTP. We further demonstrated that the arrangement of the dye residue relative to the nucleobase notably affects the outcome of the tailing reaction. A comparative study of labelled deoxycytidine and deoxyuridine nucleotides showed higher efficiency for dUTP derivatives. The non-templated addition of modified nucleotides by Taq polymerase at a duplex blunt end was generally complicated by the pyrophosphorolysis and 5' exonuclease activity of the enzyme.
非校对 DNA 聚合酶的转移酶活性是一种众所周知的现象，已被应用于克隆和测序应用中。在 DNA 平末端处非模板添加修饰核苷酸是 DNA 聚合酶的一个潜在有用的特征，可用于选择性转化 DNA 3'末端。在本文中，我们用 Cy3 和 Cy5 修饰的嘧啶核苷酸表征了完全匹配和不匹配的双链末端的加尾反应。结果表明，最佳的 DNA 加尾底物在末端没有完美的 Watson-Crick 碱基对。带有 3' dC 的不匹配双链在 Taq DNA 聚合酶催化的带有 Cy5 修饰的 dUTP 的加尾反应中是最有效的。我们进一步证明，染料残基相对于核碱基的排列显著影响加尾反应的结果。标记的脱氧胞苷和脱氧尿苷核苷酸的比较研究表明，dUTP 衍生物的效率更高。Taq 聚合酶在双链平末端的非模板修饰核苷酸的添加通常受到酶的焦磷酸解和 5'外切核酸酶活性的复杂化。
