Thermostable flap endonuclease from the archaeon, Pyrococcus horikoshii, cleaves the replication fork-like structure endo/exonucleolytically.
The flap endonuclease gene homologue from the hyperthermophilic archaeon, Pyrococcus horikoshii, was overexpressed in Escherichia coli and purified. The results of gel filtration indicated that this protein was a 41-kDa monomer. P. horikoshii flap endonuclease (phFEN) cleaves replication fork-like substrates (RF) and 5' double-strand flap structures (DF) using both flap endonuclease and 5'-3'-exonuclease activities. The mammalian flap endonuclease (mFEN) is a single-strand flap-specific endonuclease (Harrington, J. J., and Lieber, M. R. (1994) EMBO J. 13, 1235-1246), but the action patterns of phFEN appear to be quite different from those of mFEN at this point. The DF-specific flap endonuclease and 5'-exonuclease activities have not yet been reported. Therefore, this is the first report of the specific endo/exonuclease activities of phFEN. The DF-specific 5'-exonuclease activity degraded the downstream primer of 3' single-flap structure and was 15 times higher than the activities against nicked substrates without 3' flap strand. DF-specific flap endonuclease cleaved the 5' double-flap strand in DF and the lagging strand in RF at the junction portion. Because the RF appears to be the intermediate structure, due to the arrest of the replication fork, the double strand breaks after the arrests of the replication forks are probably caused by phFEN.
Matsui
E
E
National Institute of Bioscience and Human Technology, Higashi 1-1, Tsukuba, Ibaraki 305, Japan. imatsui@ccmail.nibh.go.jp
Kawasaki
S
S
Ishida
H
H
Ishikawa
K
K
Kosugi
Y
Y
Kikuchi
H
H
Kawarabayashi
Y
Y
Matsui
I
I
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004260:DNA Repair; D019641:DNA, Archaeal / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli; D045585:Flap Endonucleases; D006801:Humans; D051379:Mice; D008956:Models, Chemical; D008969:Molecular Sequence Data; D019711:Pyrococcus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
D019641:DNA, Archaeal; D011994:Recombinant Proteins; D004706:Endodeoxyribonucleases; D045585:Flap Endonucleases; C468317:FEN1 protein, human