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Imaging DNA loops induced by restriction endonuclease EcoRII. A single amino acid substitution uncouples target recognition from cooperative DNA interaction and cleavage

Item Type:Article
Title:Imaging DNA loops induced by restriction endonuclease EcoRII. A single amino acid substitution uncouples target recognition from cooperative DNA interaction and cleavage
Creators Name:Muecke, M. and Lurz, R. and Mackeldanz, P. and Behlke, J. and Krueger, D.H. and Reuter, M.
Abstract:EcoRII is a type IIE restriction endonuclease characterized by a highly cooperative reaction mechanism that depends on simultaneous binding of the dimeric enzyme molecule to two copies of its DNA recognition site. Transmission electron microscopy provided direct evidence that EcoRII mediates loop formation of linear DNA containing two EcoRII recognition sites. Specific DNA binding of EcoRII revealed a symmetrical DNase I footprint occupying 16-18 bases. Single amino acid replacement of Val258 by Asn yielded a mutant enzyme that was unaffected in substrate affinity and DNase I foot-printing properties, but exhibited a profound decrease in cooperative DNA binding and cleavage activity. Because the electrophoretic mobility of the mutant enzyme-DNA complexes was significantly higher than that of the wild-type, we investigated if mutant V258N binds as a monomer to the substrate DNA. Analysis of the molecular mass of mutant V258N showed a high percentage of protein monomers in solution. The dissociation constant of mutant V258N confirmed a 350-fold decrease of the enzyme dimerization capability. We conclude that Val258 is located in a region of EcoRII involved in homodimerization. This is the first report of a specific amino acid replacement in a restriction endonuclease leading to the loss of dimerization and DNA cleavage while retaining specific DNA binding.
Keywords:Asparagine, Deoxyribonuclease I, DNA, DNA-Binding Proteins, DNA Footprinting, Molecular Weight, Mutation, Nucleic Acid Conformation, Protein Binding, Quaternary Protein Structure, Site-Directed Mutagenesis, Type II Site-Specific Deoxyribonucleases, Ultracentrifugation, Valine
Source:Journal of Biological Chemistry
ISSN:0021-9258
Publisher:American Society for Biochemistry and Molecular Biology (U.S.A.)
Volume:275
Number:39
Page Range:30631-30637
Date:1 January 2000
Official Publication:https://doi.org/10.1074/jbc.M003904200
PubMed:View item in PubMed

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