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J Biol Chem, Vol. 274, Issue 15, 10235-10243, April 9, 1999
From the PI-SceI is an intein-encoded protein
that belongs to the LAGLIDADG family of homing endonucleases. According
to the crystal structure and mutational studies, this endonuclease
consists of two domains, one responsible for protein splicing, the
other for DNA cleavage, and both presumably for DNA binding. To define
the DNA binding site of PI-SceI, photocross-linking was
used to identify amino acid residues in contact with DNA. Sixty-three
double-stranded oligodeoxynucleotides comprising the minimal
recognition sequence and containing single 5-iodopyrimidine
substitutions in almost all positions of the recognition sequence were
synthesized and irradiated in the presence of PI-SceI with
a helium/cadmium laser (325 nm). The best cross-linking yield
(approximately 30%) was obtained with an oligodeoxynucleotide with a
5-iododeoxyuridine at position +9 in the bottom strand. The subsequent
analysis showed that cross-linking had occurred with amino acid
His-333, 6 amino acids after the second LAGLIDADG motif. With the H333A
variant of PI-SceI or in the presence of excess unmodified
oligodeoxynucleotide, no cross-linking was observed, indicating the
specificity of the cross-linking reaction. Chemical modification of His
residues in PI-SceI by diethylpyrocarbonate leads to a
substantial reduction in the binding and cleavage activity of
PI-SceI. This inactivation can be suppressed by substrate
binding. This result further supports the finding that at least one His
residue is in close contact to the DNA. Based on these and published
results, conclusions are drawn regarding the DNA binding site of
PI-SceI.
Photocross-linking of the Homing Endonuclease PI-SceI
to Its Recognition Sequence
,,,, and
Institut für Biochemie,
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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