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.

School of Biological Sciences University of the Punjab Lahore 54590 Pakistan

Zobrazit více v PubMed

Zebrafish. 2004;1(3):273-85 PubMed

Nature. 1970 Aug 15;227(5259):680-5 PubMed

Biosci Biotechnol Biochem. 2007 Apr;71(4):855-65 PubMed

J Biol Chem. 2010 Nov 5;285(45):34922-31 PubMed

Cell. 2011 Apr 15;145(2):198-211 PubMed

J Bacteriol. 2017 Jun 13;199(13): PubMed

J Biol Chem. 2002 Oct 4;277(40):37840-7 PubMed

Gene. 2003 Sep 18;314:63-71 PubMed

J Biol Chem. 2002 Apr 26;277(17):14379-89 PubMed

Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011 Feb 1;67(Pt 2):209-13 PubMed

Cell. 2004 Jan 9;116(1):39-50 PubMed

Acta Crystallogr D Biol Crystallogr. 2004 Sep;60(Pt 9):1674-8 PubMed

Cell. 1998 Oct 2;95(1):135-46 PubMed

J Mol Biol. 2003 May 2;328(3):537-54 PubMed

Extremophiles. 2014 Mar;18(2):415-27 PubMed

EMBO J. 1997 Jun 2;16(11):3341-8 PubMed

J Biol Chem. 1996 Apr 19;271(16):9173-6 PubMed

Genome Res. 2005 Mar;15(3):352-63 PubMed

Mol Cell Biol. 2005 Aug;25(16):6948-55 PubMed

J Biol Chem. 1999 Jun 25;274(26):18297-309 PubMed

J Bacteriol. 1998 Oct;180(20):5406-12 PubMed

EMBO J. 2012 Mar 21;31(6):1556-67 PubMed

Biochem J. 2009 Mar 1;418(2):285-92 PubMed

J Biol Chem. 1998 Oct 16;273(42):27154-61 PubMed

Genet Mol Res. 2003 Dec 30;2(4):383-93 PubMed

PLoS One. 2011 Mar 01;6(3):e17581 PubMed

J Mol Biol. 2007 Aug 3;371(1):34-48 PubMed

Nucleic Acids Res. 1998 Aug 15;26(16):3645-50 PubMed

J Biol Chem. 2004 Apr 16;279(16):16687-96 PubMed

J Biol Chem. 2004 Apr 9;279(15):15014-24 PubMed

J Biol Chem. 1997 Feb 21;272(8):4647-50 PubMed

Appl Environ Microbiol. 1994 Dec;60(12):4559-66 PubMed

Archaea. 2004 Oct;1(4):263-7 PubMed