NEWLY DISCOVERED ARCHAEBACTERIAL FLAP ENDONUCLEASES SHOW A STRUCTURE-SPECIFIC MECHANISM FOR DNA SUBSTRATE-BINDING AND CATALYSIS RESEMBLING HUMAN FLAP ENDONUCLEASE-1

Mammalian flap endonuclease-l (FEN-1) is a structure-specific metalloe nzyme that acts in processing of both the Okazaki fragments during lag ging strand DNA synthesis and flap intermediates during DNA damage rep air. We identified and cloned three open reading frames encoding a fla p endonuclease from Archaeglobus fulgidus,,Methanococcus jannaschii, a nd Pyrococcus furiosus, respectively. The deduced FEN-1 protein sequen ces share approximately 75% similarity with the human FEN-1 nuclease i n the conserved nuclease domains, and extensive biochemical experiment s indicate that the substrate specificities and catalytic activities o f these enzymes have overall similarities with those of the human enzy me, Thus, FEN-1 enzymes and likely reaction mechanisms are conserved a cross the eukaryotic and archaeal kingdoms. Detailed comparative analy sis, however, reveals subtle differences among these four enzymes incl uding distinctive substrate specificity, tolerance of the archaebacter ial enzymes for acidic pHs and elevated temperatures, and variations i n the metalion dependence of substrate cleavage. Although the archaeba cterial enzymes were inactive at temperatures below 30 degrees C, DNA binding occurred at temperatures as low as 4 degrees C and with or wit hout metal ions. Thus, these archaeal enzymes mag provide a means to d issect the specific binding and catalytic mechanisms of the entire FEN -1 family of structure-specific nucleases.