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

EcoRII is a type IIE restriction endonuclease characterized by a highly coo perative reaction mechanism that depends on simultaneous binding of the dim eric enzyme molecule to two copies of its DNA recognition site. Transmissio n electron microscopy provided direct evidence that EcoRII mediates loop fo rmation of linear DNA containing two EcoRII. recognition sites. Specific DN A binding of EcoRII revealed a symmetrical DNase I footprint occupying 16-1 8 bases. Single amino acid replacement of Val(258) by Asn yielded a mutant enzyme that was unaffected in substrate affinity and DNase I footprinting p roperties, but exhibited a profound decrease in cooperative DNA binding and cleavage activity. Because the electrophoretic mobility of the mutant enzy me-DNA complexes was significantly higher than that of the wild-type, we in vestigated if mutant V258N binds as a monomer to the substrate DNA. Analysi s 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 Val(258) is located in a region of EcoRII involved in homodimerization. Th is is the first report of a specific amino acid replacement in a restrictio n endonuclease leading to the loss of dimerization and DNA cleavage while r etaining specific DNA binding.