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Journal of Bacteriology, October 1998, p. 5406-5412, Vol. 180, No. 20
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Methanococcus jannaschii Flap Endonuclease: Expression, Purification, and Substrate Requirements

H. G. V. Rao, Amy Rosenfeld, and James G. Wetmur*

Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029

Received 12 March 1998/Accepted 7 August 1998

The flap endonuclease (FEN) of the hyperthermophilic archaeon Methanococcus jannaschii was expressed in Escherichia coli and purified to homogeneity. FEN retained activity after preincubation at 95°C for 15 min. A pseudo-Y-shaped substrate was formed by hybridization of two partially complementary oligonucleotides. FEN cleaved the strand with the free 5' end adjacent to the single-strand-duplex junction. Deletion of the free 3' end prevented cleavage. Hybridization of a complementary oligonucleotide to the free 3' end moved the cleavage site by 1 to 2 nucleotides. Hybridization of excess complementary oligonucleotide to the free 5' end failed to block cleavage, although this substrate was refractory to cleavage by the 5'-3' exonuclease activity of Taq DNA polymerase. For verification, the free 5' end was replaced by an internally labeled hairpin structure. This structure was a substrate for FEN but became a substrate for Taq DNA polymerase only after exonucleolytic cleavage had destabilized the hairpin. A circular duplex substrate with a 5' single-stranded branch was formed by primer extension of a partially complementary oligonucleotide on virion phi X174. This denaturation-resistant substrate was used to examine the effects of temperature and solution properties, such as pH, salt, and divalent ion concentration on the turnover number of the enzyme.

* Corresponding author. Mailing address: Department of Microbiology, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029-6574. Phone: (212) 241-7685. Fax: (212) 534-1684. E-mail: wetmur{at}msvax.mssm.edu.

Journal of Bacteriology, October 1998, p. 5406-5412, Vol. 180, No. 20
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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