title: Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV.
abstract: The DNA binding stringency of restriction endonucleases is crucial for their proper function. The X-ray structures of the specific and non-cognate complexes of the restriction nuclease EcoRV are considerably different suggesting significant differences in the hydration and binding free energies. Nonetheless, the majority of studies performed at pH 7.5, optimal for enzymatic activity, have found a < 10-fold difference between EcoRV binding constants to the specific and nonspecific sequences in the absence of divalent ions. We used a recently developed self-cleavage assay to measure EcoRV-DNA competitive binding and to evaluate the influence of water activity, pH and salt concentration on the binding stringency of the enzyme in the absence of divalent ions. We find the enzyme can readily distinguish specific and nonspecific sequences. The relative specific-nonspecific binding constant increases strongly with increasing neutral solute concentration and with decreasing pH. The difference in number of associated waters between specific and nonspecific DNA-EcoRV complexes is consistent with the differences in the crystal structures. Despite the large pH dependence of the sequence specificity, the osmotic pressure dependence indicates little change in structure with pH. The large osmotic pressure dependence means that measurement of protein-DNA specificity in dilute solution cannot be directly applied to binding in the crowded environment of the cell. In addition to divalent ions, water activity and pH are key parameters that strongly modulate binding specificity of EcoRV.
authors.affiliation: Laboratory of Physical and Structural Biology, Program of Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-0924, USA. sidorova@mail.nih.gov
authors.forename: Nina Y
authors.initials: NY
authors.lastname: Sidorova
authors.forename: Shakir
authors.initials: S
authors.lastname: Muradymov
authors.forename: Donald C
authors.initials: DC
authors.lastname: Rau
mesh_terms: D001667:Binding, Competitive; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009997:Osmotic Pressure; D011485:Protein Binding / Q000187:drug effects; D012492:Salts; D012996:Solutions; D013379:Substrate Specificity; D014867:Water / Q000737:chemistry
chemical_list: D012492:Salts; D012996:Solutions; D014867:Water; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases