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J Biol Chem, Vol. 275, Issue 4, 2705-2712, January 28, 2000
From the The PI-SceI protein is an
intein-encoded homing endonuclease that initiates the mobility of its
gene by making a double strand break at a single site in the yeast
genome. The PI-SceI protein splicing and endonucleolytic
active sites are separately located in each of two domains in the
PI-SceI structure. To determine the spatial relationship
between bases in the PI-SceI recognition sequence and
selected PI-SceI amino acids, the PI-SceI-DNA
complex was probed by photocross-linking and affinity cleavage methods. Unique solvent-accessible cysteine residues were introduced into the
two PI-SceI domains at positions 91, 97, 170, 230, 376, and 378, and the mutant proteins were modified with either 4-azidophenacyl bromide or iron
(S)-1-(p-bromoacetamidobenzyl)-ethylenediaminetetraacetate (FeBABE). The phenyl azide-coupled proteins cross-linked to the PI-SceI target sequence, and the FeBABE-modified proteins
cleaved the DNA proximal to the derivatized amino acid. The results
suggest that an extended The atomic coordinates (code 1DFA) have been deposited in the
Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
Probing the Structure of the PI-SceI-DNA Complex by
Affinity Cleavage and Affinity Photocross-linking*
,,
Center for Genome Research, Institute of
Biosciences and Technology and Department of Medical Biochemistry and
Genetics, The Texas A & M University System Health Science Center,
Houston, Texas 77030 and the § Howard Hughes Medical
Institute and Verna Marrs McLean Department of Biochemistry and
Molecular Biology, Baylor College of Medicine,
Houston, Texas 77030
-hairpin loop in the endonuclease domain
that contains residues 376 and 378 contacts the major groove near the PI-SceI cleavage site. Conversely, residues 91, 97, and 170 in the protein splicing domain are in close proximity to a distant region of the substrate. To interpret our results, we used a new PI-SceI structure that is ordered in regions of the protein
that bind DNA. The data strongly support a model of the
PI-SceI-DNA complex derived from this structure.
*
This work was supported by National Institutes of Health
Grant GM50815 (to F. S. G.) and by funds from the Institute
of Biosciences and Technology (F. S. G.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Center for Genome
Research, Institute of Biosciences and Technology, 2121 W. Holcombe Blvd., Houston, TX 77030. Tel.: 713-677-7605; Fax: 713-677-7689; E-mail: fgimble@ibt.tamu.edu.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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