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 Val(258) by Asn yielded a mutant enzyme that was unaffected in substrate affinity and DNase I footprinting 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 Val(258) 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.
Highlights
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
We examined the role of single amino acid residues of EcoRII DNA-binding region II in targetsite recognition and catalytic activity
EcoRII exists as a homodimer in solution but, unlike most type II ENases, must bind two recognition sites simultaneously, either in cis or in trans, for effective DNA cleavage (1)
Summary
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 cooperative reaction mechanism that depends on simultaneous binding of the dimeric enzyme molecule to two copies of its DNA recognition site. Single amino acid replacement of Val258 by Asn yielded a mutant enzyme that was unaffected in substrate affinity and DNase I footprinting properties, but exhibited a profound decrease in cooperative DNA binding and cleavage activity. 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. No cleavage occurred when the spacing between two sites exceeded a critical length of 1000 bp Overall, these biochemical studies suggest that EcoRII takes advantage of DNA bending or looping to achieve cooperativity in cis (11–14). The experimental data confirm the proposed two-site cooperative reaction mechanism of EcoRII
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