Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.
PLoS One. 2013 Aug 5;8(8):e70892. doi: 10.1371/journal.pone.0070892. Print 2013.
Thermophilic DNA polymerases of the polB family are of great importance in biotechnological applications including high-fidelity PCR. Of particular interest is the relative promiscuity of engineered versions of the exo- form of polymerases from the Thermo- and Pyrococcales families towards non-canonical substrates, which enables key advances in Next-generation sequencing. Despite this there is a paucity of structural information to guide further engineering of this group of polymerases. Here we report two structures, of the apo form and of a binary complex of a previously described variant (E10) of Pyrococcus furiosus (Pfu) polymerase with an ability to fully replace dCTP with Cyanine dye-labeled dCTP (Cy3-dCTP or Cy5-dCTP) in PCR and synthesise highly fluorescent "CyDNA" densely decorated with cyanine dye heterocycles. The apo form of Pfu-E10 closely matches reported apo form structures of wild-type Pfu. In contrast, the binary complex (in the replicative state with a duplex DNA oligonucleotide) reveals a closing movement of the thumb domain, increasing the contact surface with the nascent DNA duplex strand. Modelling based on the binary complex suggests how bulky fluorophores may be accommodated during processive synthesis and has aided the identification of residues important for the synthesis of unnatural nucleic acid polymers.
嗜热 DNA 聚合酶的 polB 家族在生物技术应用中具有重要意义，包括高保真 PCR。特别有趣的是，来自 Thermo 和 Pyrococcales 家族的 exo-形式聚合酶的工程版本对非规范底物的相对混杂性，这使得新一代测序技术取得了关键进展。尽管如此，关于这个聚合酶家族的结构信息仍然很少，无法进一步进行工程设计。在这里，我们报告了两种结构，一种是 apo 形式，另一种是先前描述的 Pyrococcus furiosus (Pfu) 聚合酶变体 (E10) 的二元复合物的结构，该变体能够在 PCR 中完全用 Cy3-dCTP 或 Cy5-dCTP 取代 dCTP，合成高度荧光的“CyDNA”，并密集地修饰有氰基染料杂环。Pfu-E10 的 apo 形式与报道的野生型 Pfu 的 apo 形式结构非常匹配。相比之下，二元复合物（在具有双链 DNA 寡核苷酸的复制状态下）揭示了拇指结构域的闭合运动，增加了与新生 DNA 双链链的接触表面。基于二元复合物的建模表明，在连续合成过程中如何容纳大体积的荧光团，并有助于鉴定对合成非天然核酸聚合物重要的残基。
