Homing Endonuclease and Protein Splicing Activity of Inteins from Extreme Thermophiles

Abstract

Protein splicing is a post‐translational modification by which intervening polypeptides, or inteins, catalyze their own excision from two flanking polypeptides, or exteins, concomitant with the ligation of the exteins. Many inteins contain a homing endonuclease domain, which can facilitate intein homing by catalyzing double strand breaks in intein‐less extein alleles. We studied the in vitro protein splicing and endonuclease activity of homing endonuclease‐containing inteins that interrupt the DNA Polymerase II (PolII) from the deep‐sea thermophiles Thermococcus kodakarensis (Tko) and Thermococcus barophilus (Tba). It was found that the Tba and Tko inteins can be purified as unspliced precursors and induced to splice in vitro at temperatures greater than 37°C. Additionally, when incubated at pressures up to 1500atm, the Tba and Tko inteins continue to exhibit splicing activity. Tba and Tko inteins also have temperature‐dependent endonuclease activity; the Tba intein is enzymatically active at pressures up to 1000atm. Future work will focus on identifying the active site residues of the homing endonuclease within the Tba and Tko inteins as well as determining the target sequence specificity of the enzyme. We also will determine if there is a difference in homing endonuclease activity between excised inteins and unsplicing intein precursors.Support or Funding InformationThis work was supported by the National Science Foundation (grant MCB‐1517138) and by the Camille & Henry Dreyfus Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.