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

Kayoko Komori, Naoki Fujita, Kenji Ichiyanagi, Hideo Shinagawa, Kosuke Morikawa, Yoshizumi Ishino

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

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-PfuI and PI-PfuII. 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 MgCl2, have been determined. The high affinity for junction DNA of PI-PfuI was confirmed using the purified protein.

Original languageEnglish
Pages (from-to)4167-4174
Number of pages8
JournalNucleic acids research
Volume27
Issue number21
DOIs
Publication statusPublished - Nov 1 1999

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Pyrococcus furiosus
Inteins
Endonucleases
Proteins
DNA
Cruciform DNA
Ribonucleotide Reductases
DNA Cleavage
Magnesium Chloride
Protein Precursors
Deoxyribonuclease I
Archaea
Genes
Organism Cloning
Amino Acid Sequence
Molecular Weight
Alleles
Phosphates
PI-PfuI intein
Peptides

All Science Journal Classification (ASJC) codes

  • Genetics

Cite this

PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. I. Purification and identification of the homing-type endonuclease activities. / Komori, Kayoko; Fujita, Naoki; Ichiyanagi, Kenji; Shinagawa, Hideo; Morikawa, Kosuke; Ishino, Yoshizumi.

In: Nucleic acids research, Vol. 27, No. 21, 01.11.1999, p. 4167-4174.

Research output: Contribution to journalArticle

Komori, Kayoko ; Fujita, Naoki ; Ichiyanagi, Kenji ; Shinagawa, Hideo ; Morikawa, Kosuke ; Ishino, Yoshizumi. / PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. I. Purification and identification of the homing-type endonuclease activities. In: Nucleic acids research. 1999 ; Vol. 27, No. 21. pp. 4167-4174.
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