Using the osmotic stress technique together with a self-cleavage assay we measure directly differences in sequestered water between specific and nonspecific DNA-BamHI complexes as well as the numbers of water molecules released coupled to specific complex formation. The difference between specific and nonspecific binding free energy of the BamHI scales linearly with solute osmolal concentration for seven neutral solutes used to set water activity. The observed osmotic dependence indicates that the nonspecific DNA-BamHI complex sequesters some 120 -150 more water molecules than the specific complex. The weak sensitivity of the difference in number of waters to the solute identity suggests that these waters are sterically inaccessible to solutes. This result is in close agreement with differences in the structures determined by x-ray crystallography. We demonstrate additionally that when the same solutes that were used in competition experiments are used to probe changes accompanying the binding of free BamHI to its specific DNA sequence, the measured number of water molecules released in the binding process is strikingly solute-dependent (with up to 10-fold difference between solutes). This result is expected for reactions resulting in a large change in a surface exposed area.Whereas it is generally accepted that hydration water plays an important role in DNA-protein sequence-specific recognition (1) there are only few techniques available to probe its contribution reliably. We use an approach termed the osmotic stress technique (2, 3) that measures changes in hydration coupled with changes in the functional state by measuring the effect of water activity on reaction equilibria and kinetics. The osmotic stress technique has been used previously to measure the changes in hydration accompanying the DNA binding of several regulatory proteins: Escherichia coli gal (4), lac (5), tyr (6), and Cro (7) repressors, E. coli CAP protein (8), Hin recombinase (9), Ultrathorax and Deformed homeodomains (10), the restriction endonucleases , BamHI (15,16), and EcoRV (17), HhaI methyltransferase (18), Sso7d protein (19), the TATA-binding protein (20,21), and the E2C protein from papillomavirus (22).The dependence of an equilibrium constant on the bulk solution osmotic pressure that is varied by adding neutral solutes that do not bind directly to the DNA or protein gives a difference in the number of associated waters between the initial reactants and the final products. More precisely, water is considered associated with the DNA, protein, and complex if it excludes osmolyte, otherwise there is no osmotic imbalance. Obviously, water that is sterically sequestered in cavities, channels, or pockets fulfills this requirement. All solutes that are excluded from these cavities act identically on the equilibrium. Water molecules hydrating exposed macromolecular surfaces are more problematic. For the most part, solutes are excluded from these waters due to "preferential hydration," macromolecules prefer their interactions with water over...

Summary 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 less than 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. In spite of 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.

We take advantage of our previous observation that neutral osmolytes can strongly slow down the rate of DNA–protein complex dissociation to develop a method that uses osmotic stress to ‘freeze’ mixtures of DNA–protein complexes and prevent further reaction enabling analysis of the products. We apply this approach to the gel mobility shift assay and use it to modify a self-cleavage assay that uses the nuclease activity of the restriction endonucleases to measure sensitively their specific binding to DNA. At sufficiently high concentrations of neutral osmolytes the cleavage reaction can be triggered at only those DNA fragments with initially bound enzyme. The self-cleavage assay allows measurement of binding equilibrium and kinetics directly in solution avoiding the intrinsic problems of gel mobility shift and filter binding assays while providing the same sensitivity level. Here we compare the self-cleavage and gel mobility shift assays applied to the DNA binding of EcoRI and BamHI restriction endonucleases. Initial results indicate that BamHI dissociation from its specific DNA sequence is strongly linked to water activity with the half-life time of the specific complex increasing ∼20-fold from 0 to 1 osmolal betaine.