title: Thermodynamic and kinetic basis for the relaxed DNA sequence specificity of "promiscuous" mutant EcoRI endonucleases.
abstract: Promiscuous mutant EcoRI endonucleases produce lethal to sublethal effects because they cleave Escherichia coli DNA despite the presence of the EcoRI methylase. Three promiscuous mutant forms, Ala138Thr, Glu192Lys and His114Tyr, have been characterized with respect to their binding affinities and first-order cleavage rate constants towards the three classes of DNA sites: specific, miscognate (EcoRI*) and non-specific. We have made the unanticipated and counterintuitive observations that the mutant restriction endonucleases that exhibit relaxed specificity in vivo nevertheless bind more tightly than the wild-type enzyme to the specific recognition sequence in vitro, and show even greater preference for binding to the cognate GAATTC site over miscognate sites. Binding preference for EcoRI* over non-specific DNA is also improved. The first-order cleavage rate constants of the mutant enzymes are normal for the cognate site GAATTC, but are greater than those of the wild-type enzyme at EcoRI* sites. Thus, the mutant enzymes use two mechanisms to partially bypass the multiple fail-safe mechanisms that protect against cleavage of genomic DNA in cells carrying the wild-type EcoRI restriction-modification system: (a) binding to EcoRI* sites is more probable than for wild-type enzyme because non-specific DNA is less effective as a competitive inhibitor; (b) the combination of increased affinity and elevated cleavage rate constants at EcoRI* sites makes double-strand cleavage of these sites a more probable outcome than it is for the wild-type enzyme. Semi-quantitative estimates of rates of EcoRI* site cleavage in vivo, predicted using the binding and cleavage constants measured in vitro, are in accord with the observed lethal phenotypes associated with the three mutations.
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Sapienza
authors.forename: Crystal A
authors.initials: CA
authors.lastname: Dela Torre
authors.forename: William H
authors.initials: WH
authors.lastname: McCoy
authors.forename: Samyukta V
authors.initials: SV
authors.lastname: Jana
authors.forename: Linda
authors.initials: L
authors.lastname: Jen-Jacobson
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D019295:Computational Biology; D004247:DNA / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015246:Deoxyribonuclease EcoRI / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D004247:DNA; D015246:Deoxyribonuclease EcoRI