Nucleic Acids Research, Vol 27, Issue 21 4175-4182, Copyright © 1999 by Oxford University Press
K Komori, K Ichiyanagi, K Morikawa and Y Ishino
PI- Pfu I and PI- Pfu II from Pyrococcus furiosus are homing endonucleases,
as shown in the accompanying paper. These two endonucleases are produced by
protein splicing from the precursor protein including ribonucleotide
reductase (RNR). We show here that both enzymes specifically interact with
their substrate DNA and distort the DNA strands by 73 degrees and 67
degrees, respectively. They have two copies of the amino acid sequence
motif LAGLIDADG, which is present in the majority of homing endonucleases
and provides some of the catalytic residues necessary for DNA cleavage
activity. Site-specific mutagenesis studies showed that two acidic residues
in the motifs, Asp149 and Glu250 in PI- Pfu I, and Asp156 and Asp249 in PI-
Pfu II, were critical for catalysis. The third residues of the active site
triads, as predicted from the structure of PI- Sce I, were Asn225 in PI-
Pfu I and Lys224 in PI- Pfu II. Substitution of Asn225 in PI- Pfu I by Ala
did not affect catalysis. The cleavage activity of PI- Pfu II was 50-fold
decreased by the substitution of Ala for Lys224. The binding affinity of
the mutant protein for the substrate DNA also decreased 6- fold. The Lys in
PI- Pfu II may play a direct or indirect role in catalysis of the
endonuclease activity.
ARTICLES
PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. II. Characterization Of the binding and cleavage abilities by site-directed mutagenesis
Department of Molecular Biology, Biomolecular Engineering Research Institute, Suita, Osaka, Japan.
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