Biochemical characterization of a thermostable endonuclease V from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5

Abstract

Endonuclease V (Endo V) is an important enzyme for repairing deoxyinosine in DNA. While bacterial and eukaryotic endo Vs have been well studied, knowledge of archaeal endo Vs is limited. Here, we first presented biochemical characterization of a thermostable endonuclease V from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba endo V). The recombinant enzyme possessed optimal endonuclease activity for cleaving deoxyinosine-containing DNA at 70-90 °C. Furthermore, Tba endo V can withstand 100 °C for 120 min without significant loss of its activity, suggesting the enzyme is thermostable. Tba endo V exhibited varying cleavage efficiencies at various pH levels from 6.0 to 11.0, among which an optimal pH for the enzyme was 8.0-9.0. In addition, a divalent metal ion was required for the enzyme to cleave DNA. Mn and Mg were optimal ions for the enzyme's activity whereas Ca, Zn and Co inhibited the enzyme activity. Moreover, the enzyme activity was suppressed by high NaCl concentration. Tba endo V bound to all DNA substrates; however, the enzyme exhibited a higher affinity for binding to deoxyinosine-containing DNA than normal DNA. Our work provides valuable information for revealing the role of Tba endo V in the base excision repair pathway for deoxyinosine repair in Thermococcus.

Characterization of a novel type III alcohol dehydrogenase from Thermococcus barophilus Ch5

“…The genome of T. barophilus Ch5 encodes 2,679 proteins, two thirds of which have no associated functions [24]. Furthermore, several thermostable enzymes from T. barophilus Ch5 have been characterized [25][26][27][28][29] Three putative alcohol dehydrogenases (Tba ADHs) are encoded in the genome of T. barophilus Ch5, including one short-chain ADH (GenBank: ALM74123) and two putative type III ADHs (GenBank: ALM74514 and ALM74601). Interestingly, these two putative Fe-ADHs from T. barophilus Ch5 have only 26% similarity in amino acid compositions, suggesting that they might harbor distinct characteristics.…”

Repair of Hypoxanthine in DNA Revealed by DNA Glycosylases and Endonucleases From Hyperthermophilic Archaea

“…By comparison, Afu-EndoV and Tba-EndoV act on only Hx-containing DNA (Liu et al, 2000;Wang et al, 2018). Secondly, Tba-EndoV can bind normal DNA and Hx-containing DNA, and display higher binding Hx-containing DNA (Wang et al, 2018), while Pfu-EndoV can only bind Hx-containing DNA (Kiyonari et al, 2014). Furthermore, Pfu-EndoV is inactive to cleave DNA at pH 6.0 (Kiyonari et al, 2014), while Tba-EndoV still possesses strong cleavage activity at pH 6.0 (Wang et al, 2018).…”

“…EndoV is ubiquitously present in three domains: bacteria, Archaea, and eukaryotes ( Figure 1B ). Alignment of amino acid sequences of EndoV homologs from bacteria, Archaea, and eukaryotes has shown that EndoV has seven conserved motifs ( Wang et al, 2018 ), suggesting similar catalytic mechanism.…”

Archaeal DNA Repair Mechanisms

“…The 3 unsaturated aldehyde must be chemically converted to a 3 OH by an AP endonuclease (Endo IV in T. kodakarensis) before strand-displacement synthesis by Pol B, flap cleavage by Fen1, and ligation of the resultant nick by DNA ligase. Structural studies of AGOG have also provided insight into the structural basis of specificity, determining recognition and cleavage of damaged substrates by AGOG is mediated by a conserved proline and phenylalanine motif allowing appropriate conformational freedom [103][104][105].…”