Institute for Physical-Chemical Medicine, Moscow, 119435, Russia.
W. A. Engelhardt Institute of Molecular Biology Russian Academy of Sciences, Moscow, 119991, Russia.
Sci Rep. 2017 Jul 27;7(1):6674. doi: 10.1038/s41598-017-06136-9.
Non-natural nucleotide substrates are widely used in the enzymatic synthesis of modified DNA. The terminal activity of polymerases in the presence of modified nucleotides is an important, but poorly characterized, aspect of enzymatic DNA synthesis. Here, we studied different types of polymerase activity at sequence ends using extendable and non-extendable synthetic models in the presence of the Cy5-dUTP analog Y. In primer extension reactions with selected exonuclease-deficient polymerases, nucleotide Y appeared to be a preferential substrate for non-templated 3'-tailing, as determined by MALDI mass-spectrometry and gel-electrophoresis. This result was further confirmed by the 3'-tailing of a non-extendable hairpin oligonucleotide model. Additionally, DNA polymerases induce an exchange of the 3' terminal thymidine for a non-natural nucleotide via pyrophosphorolysis in the presence of inorganic pyrophosphate. In primer extension reactions, the proofreading polymerases Vent, Pfu, and Phusion did not support the synthesis of Y-modified primer strand. Nevertheless, Pfu and Phusion polymerases were shown to initiate terminal nucleotide exchange at the template. Unlike non-proofreading polymerases, these two enzymes recruit 3'-5' exonuclease functions to cleave the 3' terminal thymidine in the absence of pyrophosphate.
非天然核苷酸底物广泛应用于修饰 DNA 的酶促合成。在修饰核苷酸存在的情况下，聚合酶的末端活性是酶促 DNA 合成的一个重要但特征描述较差的方面。在这里，我们使用可扩展和不可扩展的合成模型，在 Cy5-dUTP 类似物 Y 的存在下，研究了序列末端的不同类型聚合酶活性。在具有所选核酸外切酶缺陷聚合酶的引物延伸反应中，通过 MALDI 质谱和凝胶电泳，核苷酸 Y 似乎优先作为非模板 3' 加尾的底物。这一结果通过不可扩展的发夹寡核苷酸模型的 3' 加尾进一步得到证实。此外，在无机焦磷酸存在下，DNA 聚合酶通过焦磷酸解诱导 3' 末端胸苷与非天然核苷酸的交换。在引物延伸反应中，校对聚合酶 Vent、Pfu 和 Phusion 不支持 Y 修饰引物链的合成。然而，Pfu 和 Phusion 聚合酶被证明能够在模板上起始末端核苷酸交换。与非校对聚合酶不同，这两种酶在没有焦磷酸盐的情况下招募 3'-5' 核酸外切酶功能来切割 3' 末端胸苷。
