Abstract
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 beta-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.
Highlights
Homing endonucleases are a class of enzymes encoded by introns and inteins that initiate the mobility of their genetic elements to sites in recipient alleles where the elements are absent
The results suggest that an extended -hairpin loop in the endonuclease domain that contains residues 376 and 378 contacts the major groove near the PI-SceI cleavage site
I-CreI differs from PI-SceI in two significant respects; it lacks a protein splicing domain, and it is a homodimeric protein composed of two molecules that are related by 2-fold symmetry (14), each of which is topologically similar to the N or C subdomains of the PI-SceI endonuclease domain
Summary
Materials—Oligonucleotides used for mutagenesis were synthesized by Sigma. Restriction and DNA-modifying enzymes were obtained from New England Biolabs, Inc. The 2.4-Å PI-SceI structure of the P21 space group crystal was used without water molecules as the search model using the data from 15 to 4 Å. PI-SceI-DNA complexes were formed by incubating the modified protein derivatives (200 nM) with these labeled DNA duplexes (ϳ0.5 nM) for 20 min in the dark in a buffer containing 25 mM HEPES (pH 8.0), 100 mM KCl, and 0.1 mM EDTA. FeBABE-mediated Affinity Cleavage—Complexes of FeBABE-modified PI-SceI derivatives and the end-labeled 158- and 187-bp DNA fragments were generated as described previously (15) except that the buffer contained 10 mM HEPES (pH 8.0), 100 mM KCl, 1 mM EDTA, and 12% glycerol. The DNA was docked to the C2 space group crystal structure of PI-SceI using methods described previously (15).
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