Clustering of null mutations in the EcoRI endonuclease
| Autor: | Herbert W. Boyer, Judith A. McClarin, Patricia J. Greene, Stephen D. Yanofsky, Robert Love, John M. Rosenberg |
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| Rok vydání: | 1987 |
| Předmět: |
DNA
Bacterial Models Molecular Protein Conformation Mutant EcoRI Mutagenesis (molecular biology technique) Protein structure function Hydroxylamine medicine.disease_cause Hydroxylamines Biochemistry Deoxyribonuclease EcoRI Endonuclease chemistry.chemical_compound Bacterial Proteins Structural Biology medicine Escherichia coli Amino Acid Sequence Molecular Biology Genetics Mutation Binding Sites biology Nucleic acid sequence DNA Restriction Enzymes Molecular biology Recombinant Proteins chemistry Genes Genes Bacterial biology.protein DNA |
| Zdroj: | Proteins. 2(4) |
| ISSN: | 0887-3585 |
| Popis: | EcoRI endonuclease mutants were isolated in a methylase-deficient background following in vitro hydroxylamine mutagenesis of plasmid pKG2 (Kuhn et al.: Gene 44:253-263, 1986). Mutants which survived high-level endonuclease expression (IPTG induction) were termed null mutants. Sixty-two of 121 null mutants tested by Western blot contained normal levels of endonuclease cross-reacting protein. The complete endonuclease gene was sequenced for 27 null mutants. This group was found to consist of 20 single base-change missense mutations, 6 double mutations, and 1 triple mutation. Ten of the 20 single mutations were clustered between residues 139 and 144. When examined with respect to the structure of the EcoRI-DNA complex (McClarin et al.: Science 234:1526-1541, 1986), these alterations were found to fall predominantly into two classes: substitutions at the protein-DNA interface or substitutions at the protein-protein (dimer) interface. Protein from several of the mutants was purified and sized by using HPLC. Wild-type EcoRI endonuclease and protein from three of the DNA interface mutations (Ala139----Thr, Gly140----Ser, Arg203----Gln) appeared to be dimeric, while protein from subunit interface mutations (Glu144----Lys, Glu152----Lys, Gly210----Arg) migrated as monomers. |
| Databáze: | OpenAIRE |
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