PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. I. Purification acid identification of the homing-type endonucleaseactivities
K. Komori et al., PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. I. Purification acid identification of the homing-type endonucleaseactivities, NUCL ACID R, 27(21), 1999, pp. 4167-4174
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.