Three proteins, yeast transcription regulatory protein GCN4, M.FokI DNA methyltransferase and R.FokI restriction endonuclease (ENase) were used to attain specific cleavage of DNA at the 18-20-bp GCN4 recognition site. This is a novel version of the 'Achilles' heel cleavage' (AC) technique [Koob et al., Science 241 (1988) 1084-1086]. Since the method employs a class-IIS ENase (R.FokI), which cleaves the DNA outside of its recognition sequence, it leaves the overlapping GCN4-binding intact. Thus, the same GCN4 site can be used in consecutive cleavage reactions. This novel GCN4-IIS-AC technique was applied to study the protein-DNA interaction. Quantitative analysis of the effect of temperature, reaction time, and GCN4 and M.FokI concentrations allowed determination of the GCN4-DNA complex half-life, which was found to be 7 h at 30 degrees C, 18 h at 22 degrees C and over 24 h at 10 degrees C. In addition, conditions for controlled, partial GCN4-IIS-AC digestion of DNA were determined, and applied to the physical mapping of large genomes.
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| Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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| Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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| Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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| Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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| Site | Modification | Modifier | Source | Reference |
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| Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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| Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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| Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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| Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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