PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. I. Purification and identification of the homing-type endonuclease activities.
AUTOR(ES)
Komori, K
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=148690Documentos Relacionados
- PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. II. Characterization Of the binding and cleavage abilities by site-directed mutagenesis.
- DNA substrate specificity and cleavage kinetics of an archaeal homing-type endonuclease from Pyrobaculum organotrophum.
- The Thy Pol-2 intein of Thermococcus hydrothermalis is an isoschizomer of PI-TliI and PI-TfuII endonucleases
- Characterization of two intein homing endonucleases encoded in the DNA polymerase gene of Pyrococcus kodakaraensis strain KOD1.
- Bioenergetics of sulfur reduction in the hyperthermophilic archaeon Pyrococcus furiosus.