Mapping of a DNA binding region of the PI-sceI homing endonuclease by affinity cleavage and alanine-scanning mutagenesis.
The PI-SceI protein is a member of the LAGLIDADG family of homing endonucleases that is generated by a protein splicing reaction. PI-SceI has a bipartite domain structure, and the protein splicing and endonucleolytic reactions are catalyzed by residues in domains I and II, respectively. Structural and mutational evidence indicates that both domains mediate DNA binding. Treatment of the protein with trypsin breaks a peptide bond within a disordered region of the endonuclease domain situated between residues Val-270 and Leu-280 and interferes with the ability of this domain to bind DNA. To identify specific residues in this region that are involved in DNA binding and/or catalysis, alanine-scanning mutagenesis was used to create a set of PI-SceI mutant proteins that were assayed for activity. One of these mutants, N281A, was >300-fold less active than wild-type PI-SceI, and two other proteins, R277A and N284A, were completely inactive. These decreases in cleavage activity parallel similar decreases in substrate binding by the endonuclease domains of these mutant proteins. We mapped the approximate position of the disordered region to one of the ends of the 31 base pair PI-SceI recognition sequence using mutant proteins that were substituted with cysteine at residues Asn-274 and Glu-283 and tethered to the chemical nuclease FeBABE. These mutational and affinity cleavage data strongly support a model of PI-SceI docked to its DNA substrate that suggests that one or more residues identified here are responsible for contacting base pair A/T(-)(9), which is essential for substrate binding.
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Center for Genome Research, Institute of Biosciences and Technology, and Department of Medical Biochemistry and Genetics, Texas A&M University System Health Science Center, 2121 West Holcombe Boulevard, Houston, Texas 77030, USA.
Crist
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Duan
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Gimble
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D000409:Alanine; D000595:Amino Acid Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D008958:Models, Molecular; D016296:Mutagenesis; D006180:Proton-Translocating ATPases; D029701:Saccharomyces cerevisiae Proteins
D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; D006180:Proton-Translocating ATPases; C081687:VMA1 protein, S cerevisiae; D000409:Alanine