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.
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http://purl.uniprot.org/cit... | rdf:type | uniprot:Journal_Citation | lld:uniprot |
http://purl.uniprot.org/cit... | rdfs:comment | 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. | lld:uniprot |
http://purl.uniprot.org/cit... | skos:exactMatch | http://purl.uniprot.org/med... | lld:uniprot |
http://purl.uniprot.org/cit... | skos:exactMatch | http://purl.uniprot.org/pub... | lld:uniprot |
http://purl.uniprot.org/cit... | uniprot:name | J. Bacteriol. | lld:uniprot |
http://purl.uniprot.org/cit... | uniprot:author | Wetmur J.G. | lld:uniprot |
http://purl.uniprot.org/cit... | uniprot:author | Rao H.G. | lld:uniprot |
http://purl.uniprot.org/cit... | uniprot:author | Rosenfeld A. | lld:uniprot |
http://purl.uniprot.org/cit... | uniprot:date | 1998 | lld:uniprot |
http://purl.uniprot.org/cit... | uniprot:pages | 5406-5412 | lld:uniprot |
http://purl.uniprot.org/cit... | uniprot:title | Methanococcus jannaschii flap endonuclease: expression, purification, and substrate requirements. | lld:uniprot |
http://purl.uniprot.org/cit... | uniprot:volume | 180 | lld:uniprot |
uniprot-protein:Q58839 | uniprot:citation | http://purl.uniprot.org/cit... | lld:uniprot |
http://linkedlifedata.com/r... | rdf:object | http://purl.uniprot.org/cit... | lld:uniprot |