title: Biochemical characterization of a thermostable HNH endonuclease from deep-sea thermophilic bacteriophage GVE2.
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 Cu(2+), the GVE2 HNH endonuclease displayed cleavage activity of varied efficiency with Mn(2+), Mg(2+), Ca(2+), Fe(2+), Co(2+), Zn(2+), and Ni(2+). 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.
authors.affiliation: Marine Science & Technology Institute, Department of Environmental Science and Engineering, Yangzhou University, Yangzhou, China. lkzhang@yzu.edu.cn.|Jiangsu Key Laboratory of Marine Biotechnology, Huaihai Institute of Technology, Lianyungang, China. lkzhang@yzu.edu.cn.|State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China. lkzhang@yzu.edu.cn.|The State Key Laboratory of Virology, Wuhan, China. lkzhang@yzu.edu.cn.
authors.forename: Likui
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Marine Science & Technology Institute, Department of Environmental Science and Engineering, Yangzhou University, Yangzhou, China.
authors.forename: Yanchao
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Dandan
authors.initials: D
authors.lastname: Xu
authors.affiliation: Marine Science & Technology Institute, Department of Environmental Science and Engineering, Yangzhou University, Yangzhou, China.
authors.forename: Lixiang
authors.initials: L
authors.lastname: Yang
authors.affiliation: Marine Science & Technology Institute, Department of Environmental Science and Engineering, Yangzhou University, Yangzhou, China.
authors.forename: Kaicheng
authors.initials: K
authors.lastname: Qian
authors.affiliation: Marine Science & Technology Institute, Department of Environmental Science and Engineering, Yangzhou University, Yangzhou, China.
authors.forename: Guozhu
authors.initials: G
authors.lastname: Chang
authors.affiliation: Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China. yonggong@ihep.ac.cn.
authors.forename: Yong
authors.initials: Y
authors.lastname: Gong
authors.affiliation: Marine Science & Technology Institute, Department of Environmental Science and Engineering, Yangzhou University, Yangzhou, China.
authors.forename: Xiaojian
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Department of Biology, University of Waterloo, Waterloo, ON, Canada.
authors.forename: Kesen
authors.initials: K
authors.lastname: Ma
mesh_terms: D001435:Bacteriophages / Q000201:enzymology* / Q000302:isolation & purification; D002413:Cations, Divalent / Q000378:metabolism; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004720:Endonucleases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012623:Seawater / Q000821:virology; D012965:Sodium Chloride / Q000378:metabolism
chemical_list: D002413:Cations, Divalent; D003067:Coenzymes; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D012965:Sodium Chloride; D004247:DNA; D004720:Endonucleases
keywords: Bacteriophage; DNA cleavage; Divalent metal ion; Endonuclease; HNH motif; Thermophile