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, P yrococcus horikoshii, was overexpressed in Escherichia coli and purified, T he results of gel filtration indicated that this protein was a 41-kDa monom er. P. horikoshii flap endonuclease (phFEN) cleaves replication fork-like s ubstrates (RF) and 5' double strand flap structures (DF) using both flap en donuclease and 5'-3'-exonuclease activities. The mammalian flap endonucleas e (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 D F specific flap endonuclease ase and 5'-exonuclease activities have not yet been reported. Therefore, this is the first report of the specific endo/ex onuclease activities of phFEN. The DF-specific 5'-exonuclease activity degr aded the downstream primer of 3' single-flap structure and was 15 times hig her than the activities against nicked substrates without 3' flap strand. D F-specific flap endonuclease cleaved the 5' double-flap strand in DF and th e lagging strand in RF at the junction portion. Because the RF appears to b e the intermediate structure, due to the arrest of the replication fork, th e double strand breaks after the arrests of the replication forks are proba bly caused by phFEN.