PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. I. Purification acid identification of the homing-type endonucleaseactivities

We screened for proteins with specific binding activity to Holliday junctio n DNA from the hyperthermophilic archaeon Pyrococcus furiosus and found a p rotein that has specific affinity for DNA with a branched structure, like a three-way or four-way junction, The protein was identified as one of the t wo inteins encoded in the gene for ribonucleotide reductase (RNR) by gene c loning, These two inteins were spliced out from the precursor protein as po lypeptides with molecular weights of 53.078 and 43.976 kDa, respectively, T he amino acid sequences of these inteins have two copies of the LAGLIDADG m otif, which is found in the site-specific DNA endonucleases, The purified p roteins actually cleaved double-stranded DNA with the sequence of the intei n(-) allele, and, therefore, they were designated PI-Pful and PI-Pfull, The y generate a 4 bp 3'-OH overhang with a 5'-phosphate, like other known homi ng endonucleases originating from inteins, The optimal conditions of the DN A cleavage reaction, including temperature, pH, and concentrations of KCI a nd MgCl2, have been determined, The high affinity for junction DNA of PI-Pf ul was confirmed using the purified protein.