Gene cloning and characterization of Tk1281, a flap endonuclease 1 from Thermococcus kodakarensis.

Flap endonuclease is a structure-specific nuclease which cleaves 5'-flap of bifurcated DNA substrates. Genome sequence of Thermococcus kodakarensis harbors an open reading frame, Tk1281, exhibiting high homology with archaeal flap endonucleases 1. The corresponding gene was cloned and expressed in Escherichia coli, and the gene product was purified to apparent homogeneity. Tk1281 was a monomer of 38 kDa and catalyzed the cleavage of 5'-flap from double-stranded DNA substrate containing single-stranded DNA flap. The highest cleavage activity was observed at 80 °C and pH 7.5. Under optimal conditions, Tk1281 exhibited apparent Vmax and Km values of 278 nmol/min/mg and 37 μM, respectively, against a 54-nucleotide double-stranded substrate containing a single-stranded 5'-flap of 27 nucleotides. A unique feature of Tk1281 is its highest activation in the presence of Co2+ and no activation with Mn2+. To the best of our knowledge, this is the first cloning and characterization of a flap endonuclease from the genus Thermococcus.