Chimeric Phi29 DNA polymerase with helix-hairpin-helix motifs shows enhanced salt tolerance and replication performance.
Chimeric Phi29 DNA polymerase with helix-hairpin-helix motifs shows enhanced salt tolerance and replication performance.
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DOI：
10.1111/1751-7915.13830
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发表时间：
2021-07
影响因子：
5.7
通讯作者：
Tian H
中科院分区：
文献类型：
--
作者：
Gao Y;He Y;Chen L;Liu X;Ivanov I;Yang X;Tian H
Phi29 DNA 聚合酶 (Phi29 Pol) 已成功应用于基于 DNA 纳米球的测序、单聚合酶分子的实时 DNA 测序以及采用边合成边测序 (SBS) 方法的纳米孔测序。其中，聚合酶辅助纳米孔测序技术将核苷酸序列分析为电流变化的函数。这种基于电流的离子测序技术需要聚合酶在高盐浓度（例如 0.3 M KCl）下进行复制。尽管如此，野生型 Phi29 Pol 的耐盐性相对较低。在这里，我们将来自嗜热菌 Methanopyrus kandleri 的拓扑异构酶 V (Topo V) 的螺旋-发夹-螺旋 (HhH)2 结构域 E-L（总共 8 个重复）融合到 Phi29 Pol COOH 末端，命名为 Phi29EL DNA 聚合酶 (Phi29EL Pol)。结构域融合将整体酶复制效率提高了四倍。 Phi29EL Pol 在比 Phi29 Pol 更广泛的盐浓度范围内催化滚环复制，将 KCl 活性浓度范围扩展至 0.3 M。此外，Glu375 突变为 Ser 或 Gln 增加了 KCl 存在下 Phi29EL Pol 的活性。在这项工作中，我们通过 (HhH)2 结构域插入产生了耐盐的 Phi29 Pol 衍生物。这种插入的多重优点使其成为 Phi29 Pol 的良好替代品，特别是用于纳米孔测序或其他需要高盐浓度的情况。 1. 对于基于离子电流的纳米孔测序，需要聚合酶、解旋酶或核酸外切酶来处理盐含量相对较高的 DNA (0.2–1 M KCl)，这是获得足够的离子强度以进行核碱基识别并提高信噪比所必需的。精细的结构特征赋予 Phi29 Pol 内在链置换能力，使其成为各种测序技术的理想酶，例如单聚合酶分子的实时 DNA 测序、基于 DNA 纳米球的测序仪和采用合成法测序的纳米孔测序技术。 2. 我们通过螺旋-发夹-螺旋结构域插入实现了在 0.3 M KCl 下工作的耐盐 Phi29 Pol 衍生物。该酶还显示出改进的滚环复制效率。 3.除了耐盐性外，Phi29EL Pol的其他性能，如持续合成能力、链位移和延伸率，并没有受到域融合的太大影响。 Phi29EL Pol的综合优势使其成为Phi29 Pol的良好替代品，特别是对于纳米孔测序或其他依赖于较高盐含量的情况。
Phi29 DNA polymerase (Phi29 Pol) has been successfully applied in DNA nanoball‐based sequencing, real‐time DNA sequencing from single polymerase molecules and nanopore sequencing employing the sequencing by synthesis (SBS) method. Among these, polymerase‐assisted nanopore sequencing technology analyses nucleotide sequences as a function of changes in electrical current. This ionic, current‐based sequencing technology requires polymerases to perform replication at high salt concentrations, for example 0.3 M KCl. Nonetheless, the salt tolerance of wild‐type Phi29 Pol is relatively low. Here, we fused helix–hairpin–helix (HhH)2 domains E‐L (eight repeats in total) of topoisomerase V (Topo V) from the hyperthermophile Methanopyrus kandleri to the Phi29 Pol COOH terminus, designated Phi29EL DNA polymerase (Phi29EL Pol). Domain fusion increased the overall enzyme replication efficiency by fourfold. Phi29EL Pol catalysed rolling circle replication in a broader range of salt concentrations than did Phi29 Pol, extending the KCl concentration range for activity up to 0.3 M. In addition, the mutation of Glu375 to Ser or Gln increased Phi29EL Pol activity in the presence of KCl. In this work, we produced a salt‐tolerant Phi29 Pol derivative by means of (HhH)2 domain insertion. The multiple advantages of this insertion make it a good substitute for Phi29 Pol, especially for use in nanopore sequencing or other circumstances that require high salt concentrations. 1. For ionic current based nanopore sequencing, polymerases, helicases or exonucleases were required to process DNA with relatively higher contents of salt (0.2–1 M KCl) which is necessary to attain sufficient ionic strength for nucleobase recognition and increase the signal to noise ratio. The delicate structure features endow Phi29 Pol intrinsic strand displacement capability, making it an ideal enzyme for various sequencing technologies, for example real‐time DNA sequencing from single polymerase molecules, DNA nanoball‐based sequencers and nanopore sequencing technology employing sequencing by synthesis method. 2. We have achieved a salt tolerant Phi29 Pol derivative working at 0.3 M KCl by means of helix‐hairpin‐helix domain insertion. The enzyme also showed improved rolling circle replication efficiency. 3. Besides salt tolerance, other properties of Phi29EL Pol such as the processivity, strand displacement and extension rate were not affected much by domain fusion. The combined advantages of Phi29EL Pol makes it a good substitute of Phi29 Pol, especially for nanopore sequencing or other circumstances depend on higher contents of salt.
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影响因子：
7.2
作者：
Johansson, Erik;Dixon, Nicholas
通讯作者：
Dixon, Nicholas
影响因子：
11.4
作者：
Berman, Andrea J.;Kamtekar, Satwik;Steitz, Thomas A.
通讯作者：
Steitz, Thomas A.
影响因子：
11.4
作者：
BERNAD, A;ZABALLOS, A;BLANCO, L
通讯作者：
BLANCO, L
影响因子：
4.8
作者：
Belova, GI;Prasad, R;Slesarev, AI
通讯作者：
Slesarev, AI
DOI：
10.1073/pnas.81.17.5325
发表时间：
1984-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
BLANCO, L;SALAS, M
通讯作者：
SALAS, M