PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. I. Purification and identification of the homing-type endonuclease activities.
We screened for proteins with specific binding activity to Holliday junction DNA from the hyperthermophilic archaeon Pyrococcus furiosus and found a protein 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 two inteins encoded in the gene for ribonucleotide reductase (RNR) by gene cloning. These two inteins were spliced out from the precursor protein as polypeptides with molecular weights of 53.078 and 43.976 kDa, respectively. The amino acid sequences of these inteins have two copies of the LAGLIDADG motif, which is found in the site-specific DNA endonucleases. The purified proteins actually cleaved double-stranded DNA with the sequence of the intein(-)allele, and, therefore, they were designated PI- Pfu I and PI- Pfu II. They generate a 4 bp 3'-OH overhang with a 5'-phosphate, like other known homing endonucleases originating from inteins. The optimal conditions of the DNA cleavage reaction, including temperature, pH, and concentrations of KCl and MgCl(2), have been determined. The high affinity for junction DNA of PI- Pfu I was confirmed using the purified protein.
Komori
K
K
Department of Molecular Biology, Biomolecular Engineering Research Institute, Suita, Osaka, Japan.
Fujita
N
N
Ichiyanagi
K
K
Shinagawa
H
H
Morikawa
K
K
Ishino
Y
Y
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D015636:Magnesium Chloride / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009994:Osmolar Concentration; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011189:Potassium Chloride / Q000378:metabolism; D019154:Protein Splicing; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics; D013379:Substrate Specificity
D002413:Cations, Divalent; D004268:DNA-Binding Proteins; D010455:Peptides; D011994:Recombinant Proteins; D015636:Magnesium Chloride; D011189:Potassium Chloride; D004247:DNA; D004706:Endodeoxyribonucleases