Methanococcus jannaschii flap endonuclease: expression, purification, and substrate requirements.
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 degrees 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 phiX174. 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.
Rao
H G
HG
Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029, USA.
Rosenfeld
A
A
Wetmur
J G
JG
D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004260:DNA Repair; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D045585:Flap Endonucleases; D019766:Genes, Archaeal; D017017:Methanococcus / Q000201:enzymology / Q000235:genetics*; D009690:Nucleic Acid Conformation; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D019914:Taq Polymerase / Q000378:metabolism
D019843:Archaeal Proteins; D004277:DNA, Single-Stranded; D019914:Taq Polymerase; D004706:Endodeoxyribonucleases; D045585:Flap Endonucleases