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ORIGINAL RESEARCH article

Front. Microbiol.
Sec. Microbiotechnology
Volume 15 - 2024 | doi: 10.3389/fmicb.2024.1501020

Expression and functional study of DNA polymerases from Psychrobacillus sp. BL-248-WT-3 and FJAT-21963

Provisionally accepted
Yaping Sun Yaping Sun *Danny H. Ko Danny H. Ko Jie Gao Jie Gao Kang Fu Kang Fu Yuanchen Mao Yuanchen Mao Yun He Yun He Hui Tian Hui Tian
  • Research Institute of Tsinghua University in Shenzhen (RITS), Shenzhen, China

The final, formatted version of the article will be published soon.

    The properties of DNA polymerases isolated from thermophilic and mesophilic microorganisms, such as the thermophilic Geobacillus stearothermophilus (Bst) and mesophilic Bacillus subtilis phage (Phi29), have been widely researched. However, DNA polymerases in psychrophilic microorganisms remain poorly understood. In this study, we present for the first time the expression and functional characterization of DNA polymerases PWT-WT and FWT-WT from Psychrobacillus sp. BL-248-WT-3 and FJAT-21963. Enzymatic activity assays revealed that FWT-WT possessed strand displacement but lacked exonuclease activity and high ionic strength tolerance, whereas PWT-WT lacked all these properties. Further protein engineering and biochemical analysis identified D423 and S490 as critical mutation sites for improving strand displacement and tolerance to high ionic strength, specifically in the presence of 0 – 0.3 M potassium chloride (KCl), sodium chloride (NaCl), and potassium acetate (KAc). Three-dimensional structural analysis demonstrated that the size and the electric charge of the single-stranded DNA (ssDNA) encapsulation entrance were pivotal factors in the binding of the ssDNA template.

    Keywords: Psychrophilic polymerases, protein expression, high ionic strength tolerance, Strand displacement, Exonuclease activity

    Received: 24 Sep 2024; Accepted: 06 Nov 2024.

    Copyright: © 2024 Sun, Ko, Gao, Fu, Mao, He and Tian. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence: Yaping Sun, Research Institute of Tsinghua University in Shenzhen (RITS), Shenzhen, China

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