We are continuing to investigate DNA complexes of the type II restriction enzyme, EcoRV. DNA binding stringency is crucial for proper function of restriction enzymes . At present, however, results in literature suggest that EcoRV has unusually low sequence stringency in the absence of divalent ions. We have applied a self-cleavage assay, developed by us, 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. This technique monitors only enzymatically competent complexes of the endonuclease. It does not have the limitations of gel mobility shift assay while providing same level of sensitivity. 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 indicating that water activity and pH are key parameters that strongly modulate binding specificity of EcoRV in addition to divalent ions. 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. Importantly, the large osmotic pressure dependence we saw for the EcoRV and EcoRI restriction endonucleases as well as for gal and cro bacterial repressors means that measurement of protein-DNA specificity in dilute solution cannot be directly applied to binding in the crowed environment of the cell. The time needed to reach equilibrium depends on association and dissociation rates. We found that the EcoRV has quite unusual kinetics of specific complex formation in the absence of divalent ions that was not observed for EcoRI. A significant fraction of the total enzyme, 45%, forms enzymatically competent complexes unusually slowly, especially at pH 7.6. This novel result can be explained by a slow transition between two conformations (opened or closed) of the free enzyme in solution. We found that both specific and nonspecific DNA shifts equilibrium distribution toward opened or fast binding form of the protein playing a role of the allosteric regulator or a chaperone. We are continuing our investigation into the EcoRV structures responsible for the different kinetic classes of association. Restriction endonucleases major biological role is to protect bacteria from foreign DNA invasion. In the presence of Mg2+ ions restriction endonucleases become extremely precise molecular scissors cleaving foreign (unmethylated) DNA with exquisite specificity. The optimal enzymatic activity of EcoRV is reached at about pH 7.5. Cleavage activity decreases dramatically with decreasing pH while specific equilibrium binding and binding specificity strongly increase. We are now exploring the influence of pH and osmotic stress on Mg2+ binding to the EcoRV-DNA complex using self-cleavage assay. This technique allows us to measure Mg2+ binding on an exceptionally slow time-scale. We find that the time dependence of the fraction DNA cleaved after incubation of EcoRV-DNA complexes with Mg2+ exhibits a lag phase and cannot be fit with single exponential. The kinetics can be well fit with two exponential functions. The binding of Mg2+ to the pre-formed EcoRV-DNA complex involves at least two steps and DNA can only be cleaved after the second step. Each binding step is characterized by an uptake of about 30-40 water molecules, the binding of 1-2 Mg2+ ions, and the release of 1-2 H+ ions. We hypothesize that the two steps correspond to the separate binding of Mg2+ to the two enzyme subunits. The interplay of Mg2+ and pH might play a very important role in the survival mechanism of enteric bacteria that spends significant amount of time at very low pH conditions of the stomach. The association and dissociation kinetics of sequence specific DNA binding proteins are surprisingly complicated. It is generally thought that the sequence specific DNA binding proteins that regulate gene activity locate their target sequences by initially binding nonspecifically with subsequent one-dimensional diffusion along DNA interspersed with short hops or jumps and by direct transfer of protein from one DNA helix to another. Direct transfer is characterized by the formation of an intermediate DNA-protein-DNA ternary complex. Consequently, the dissociation rate depends on total DNA concentration. The direct transfer model is gaining popularity since protein-DNA dissociation rates are becoming more widely observed to depend on competitor DNA concentrations. Since most all specific or nonspecific complexes of these proteins with DNA entail charge-charge interactions, the salt concentration dependence of dissociation due to direct transfer and formation of a ternary DNA-protein complex should be quite different than from simple dissociation. As the salt concentration is increased, the contribution to dissociation from direct transfer should significantly decrease. This difference, however, has not been directly investigated. We have found that at near physiological NaCl concentrations the dissociation of the restriction endonuclease EcoRI from specific sequence DNA has both competitor DNA concentration dependent and independent contributions. At two-fold higher salt concentrations, only the competitor oligonucleotide concentration independent component is observed as expected for smaller salt concentration dependence of direct transfer compared to simple dissociation. Unexpectedly, however, no difference is seen in the salt concentration dependence of dissociation measured around physiological salt concentration for two concentrations of competitor oligonucleotide that show quite different apparent contributions from direct transfer. These disparate observations can be rationalized by considering the physical consequences of transient protein dissociation. After a protein dissociates from DNA fragment, for a short period of time it remains very close and has high probability to simply rebind to the same DNA. The probability that the protein will rebind after a time t is given by the first passage time distribution function that has been estimated for DNA-protein systems. During the time the protein is off the DNA, there is also a probability it will react and bind with competitor DNA. This probability will, of course, depend on competitor DNA concentration and the association rate. A calculation of the probability of reaction with a competitor oligonucleotide before the protein rebinds to the initial DNA indicates that this mechanism does fit the experimentally observed dependence of the dissociation rate on competitor concentration at physiological salt concentrations. Since the protein has dissociated from the initial DNA and no ternary complex is formed, the salt dependence will be independent of oligonucleotide concentration. The loss of oligonucleotide concentration dependence with doubling the salt concentration can also be understood within this mechanism. At physiological salt concentrations, we were able to determine previously that the protein can diffuse hundreds of base pairs along the DNA, far enough such that after rebinding the protein has a high probability to find the specific recognition sequence before dissociating again. At twice the salt concentration, however, the protein can only diffuse short distances along the DNA (tens of base pairs instead of hundreds). It is highly unlikely that the protein can find the specific sequence before dissociating again. Reaction with the competitor DNA is much more probable now at all practical oligonucleotide concentratio

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Sidorova, Nina Y; Scott, Thomas; Rau, Donald C (2013) DNA concentration-dependent dissociation of EcoRI: direct transfer or reaction during hopping. Biophys J 104:1296-303
Sidorova, Nina Y; Muradymov, Shakir; Rau, Donald C (2011) Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV. FEBS J 278:2713-27
Sidorova, Nina Y; Hung, Stevephen; Rau, Donald C (2010) Stabilizing labile DNA-protein complexes in polyacrylamide gels. Electrophoresis 31:648-53
Rau, Donald C; Sidorova, Nina Y (2010) Diffusion of the restriction nuclease EcoRI along DNA. J Mol Biol 395:408-16