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Biochemical characterization of a thermostable HNH endonuclease from deep-sea thermophilic bacteriophage GVE2

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Abstract

His-Asn-His (HNH) proteins are a very common family of small nucleic acid-binding proteins that are generally associated with endonuclease activity and are found in all kingdoms of life. Although HNH endonucleases from mesophiles have been widely investigated, the biochemical functions of HNH endonucleases from thermophilic bacteriophages remain unknown. Here, we characterized the biochemical properties of a thermostable HNH endonuclease from deep-sea thermophilic bacteriophage GVE2. The recombinant GVE2 HNH endonuclease exhibited non-specific cleavage activity at high temperature. The optimal temperature of the GVE2 HNH endonuclease for cleaving DNA was 60–65 °C, and the enzyme retained its DNA cleavage activity even after heating at 100 °C for 30 min, suggesting the enzyme is a thermostable endonuclease. The GVE2 HNH endonuclease cleaved DNA over a wide pH spectrum, ranging from 5.5 to 9.0, and the optimal pH for the enzyme activity was 8.0–9.0. Furthermore, the GVE2 HNH endonuclease activity was dependent on a divalent metal ion. While the enzyme is inactive in the presence of Cu2+, the GVE2 HNH endonuclease displayed cleavage activity of varied efficiency with Mn2+, Mg2+, Ca2+, Fe2+, Co2+, Zn2+, and Ni2+. The GVE2 HNH endonuclease activity was inhibited by NaCl. This study provides the basis for determining the role of this endonuclease in life cycle of the bacteriophage GVE2 and suggests the potential application of the enzyme in molecular biology and biotechnology.

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Acknowledgments

We thank Prof. Xiaobo Zhang (Zhejiang University, China) for providing the genomic DNA of GVE2. This work was supported by the National Natural Science Foundation of China Grant (41306131), the Natural Science Foundation Grant for College and University of Jiangsu Province, China (13KJB180029), the Provincial Natural Science Foundation Grant of Jiangsu Province, China (BK20130440), the open project of State Key Laboratory of Microbial Resources (SKLMR-20130603), the Open-end Funds of Jiangsu Key Laboratory of Marine Biotechnology, Huaihai Institute of Technology (2014HS008), and the open Research Fund Program of the State Key Laboratory of Virology of China (2016KF006) to L. Z., the National Natural Science Foundation of China Grant (41271521) to X. Z., the National Natural Science Foundation of China Grant (31270791) to Y. G., the Yangzhou University College Student Science and Technology Innovation Grant to K. Q., and the Natural Sciences and Engineering Research Council of Canada (NSERC) to K. M.

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Authors and Affiliations

  1. Marine Science & Technology Institute, Department of Environmental Science and Engineering, Yangzhou University, Yangzhou, China

    Likui Zhang, Yanchao Huang, Lixiang Yang, Kaicheng Qian, Guozhu Chang & Xiaojian Zhou

  2. Jiangsu Key Laboratory of Marine Biotechnology, Huaihai Institute of Technology, Lianyungang, China

    Likui Zhang

  3. State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

    Likui Zhang

  4. The State Key Laboratory of Virology, Wuhan, China

    Likui Zhang

  5. Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China

    Dandan Xu & Yong Gong

  6. Department of Biology, University of Waterloo, Waterloo, ON, Canada

    Kesen Ma

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  1. Likui Zhang
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  2. Yanchao Huang
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  3. Dandan Xu
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  4. Lixiang Yang
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  7. Yong Gong
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  8. Xiaojian Zhou
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Correspondence to Likui Zhang or Yong Gong.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Likui Zhang and Yanchao Huang contributed equally to this work.

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Zhang, L., Huang, Y., Xu, D. et al. Biochemical characterization of a thermostable HNH endonuclease from deep-sea thermophilic bacteriophage GVE2. Appl Microbiol Biotechnol 100, 8003–8012 (2016). https://doi.org/10.1007/s00253-016-7568-7

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