Biochemical characterization and mutational studies of endonuclease Q from the hyperthermophilic euryarchaeon Thermococcus gammatolerans

Endonuclease Q (EndoQ) can effectively cleave DNA containing deaminated base(s), thus providing a potential pathway for repair of deaminated DNA. EndoQ is ubiquitous in some Archaea, especially in Thermococcales, and in a small group of bacteria. Herein, we report biochemical characteristics of EndoQ from the hyperthermophilic euryarchaeon Thermococcus gammatolerans (Tga-EndoQ) and the roles of its six conserved residues in DNA cleavage. The enzyme can cleave uracil-, hypoxanthine-, and AP (apurinic/apyrimidinic) site-containing DNA with varied efficiencies at high temperature, among which uracil-containing DNA is its most preferable substrate. Additionally, the enzyme displays maximum cleavage efficiency at above 70 degrees C and pH 7.0 similar to 8.0. Furthermore, Tga-EndoQ still retains 85% activity after heated at 100 degrees C for 2 hrs, suggesting that the enzyme is extremely thermostable. Moreover, the Tga-EndoQ activity is independent of a divalent ion and NaCl. Mutational data demonstrate that residues E167 and H195 in Tga-EndoQ are essential for catalysis since the E167A and H195A mutants completely abolish the cleavage activity. Besides, residues S18 and R204 in Tga-EndoQ are involved in catalysis due to the reduced activities observed for the S18A and R204A mutants. Overall, our work has augmented biochemical function of archaeal EndoQ and provided insight into its catalytic mechanism.

Automated summary

This study reveals the biochemical characteristics of EndoQ from Thermococcus gammatolerans and the roles of its conserved residues in DNA cleavage. The enzyme is highly efficient in cleaving DNA with deaminated bases, especially uracil-containing DNA. It displays the maximum activity at high temperature and neutral to slightly alkaline pH, and it is extremely thermostable. Mutational analysis identifies essential and catalytic residues in the enzyme.