Nina Y. Sidorova

Diffusion of the Restriction Nuclease EcoRI along DNA

Many specific sequence DNA binding proteins locate their target sequence by first binding to DNA nonspecifically, then by linearly diffusing or hopping along DNA until either the protein dissociates from the DNA or it finds the recognition sequence. We have devised a method for measuring one-dimensional diffusion along DNA based on the ratio of the dissociation rate of protein from DNA fragments containing one specific binding site to the dissociation rate from DNA fragments containing two specific binding sites. Our extensive measurements of dissociation rates and specific-nonspecific relative binding constants of the restriction nuclease EcoRI enable us to determine the diffusion rate of nonspecifically bound protein along the DNA. By varying the distance between the two binding sites, we confirm a linear diffusion mechanism. The sliding rate is relatively insensitive to salt concentration and osmotic pressure, indicating that the protein moves smoothly along the DNA probably following the helical phosphate-sugar backbone of DNA. We calculate a diffusion coefficient for EcoRI of 3 x 10(4) bp(2) s(-)(1) EcoRI is able to diffuse approximately 150 bp, on average, along the DNA in 1 s. This diffusion rate is about 2000-fold slower than the diffusion of free protein in solution. A factor of 40-50 can be accounted for by rotational friction resulting from following the helical path of the DNA backbone. Two possibilities could account for the remaining activation energy: salt bridges between the DNA and the protein are transiently broken, or the water structure at the protein-DNA interface is disrupted as the two surfaces move past each other.

Differences in Hydration Coupled to Specific and Nonspecific Competitive Binding and to Specific DNA Binding of the Restriction Endonuclease BamHI

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.

DNA Concentration-Dependent Dissociation of EcoRI: Direct Transfer or Reaction during Hopping

Direct transfer of proteins between DNA helices is a recognized important feature of the recognition site search process. Direct transfer is characterized by a dissociation rate that depends on total DNA concentration. This is taken as evidence for the formation of an intermediate DNA-protein-DNA ternary complex. We find that the dissociation rate of EcoRI-DNA-specific complexes at 80 mM NaCl depends on the concentration of competitor oligonucleotide suggesting that direct transfer contributes to EcoRI dissociation. This dependence on competitor DNA concentration is not seen at 180 mM salt. A careful examination of the salt concentration dependence of the dissociation rate, however, shows that the predictions for the formation of a ternary complex are not observed experimentally. The findings can be rationalized by considering that just after dissociating from a DNA fragment the protein remains in close proximity to that fragment, can reassociate with it, and diffuse back to the recognition site rather than bind to an oligonucleotide in solution, a hopping excursion. The probability that a protein will bind to an oligonucleotide during a hop can be approximately calculated and shown to explain the data. A dependence of the dissociation rate of a DNA-protein complex on competitor DNA concentration does not necessarily mean direct transfer.

The dissociation rate of the EcoRI-DNA-specific complex is linked to water activity.

In many respects, the dissociation rate constant of a DNA– protein or protein–protein complex is as important a physical parameter as the equilibrium constant. The regulation of most cellular activities and developmental control are dynamic rather than static processes. With many techniques, the successful physicochemical characterization of a complex depends critically on the lifetime of the complex during isolation or measurement. With present technologies, the very powerful, single molecule methods used for mapping the kinetic barriers of complex dissociation reactions require lifetimes on the order of minutes. We report here that the dissociation rate of the specific complex between the restriction nuclease EcoRI and its recognition DNA sequence is strongly dependent on water activity (in addition to its known dependence on salt activity). This observation means that the dissociation rate of complexes in the crowded conditions found within cells cannot be straightforwardly predicted from dilute solution measurements, even though salt, temperature, and pH conditions are fixed to those found in vivo. In addition, these results suggest a practical method to extend the lifetime of “weak” complexes sufficiently to perform biophysical and biochemical characterizations. The thermodynamic analysis of protein, peptide, and drug interactions with DNA has focused on the sensitivity of free energies to temperature, pH, and salt concentration (reviewed in Refs. 8–11). However, the displacement of water that should accompany specific complex formation as direct DNA–protein contacts replace DNA–water and protein–water interactions (reviewed in Refs. 12 and 13) means that binding energies will also depend on water activity. The number of water molecules released to the bulk solution in the process of DNA–protein complex formation can be measured from the sensitivity of the binding constant to bulk solution water activity. This procedure is analogous to measuring ion release through the dependence of binding constant on salt activity, or protonation through pH sensitivity. Water activity can be varied by adding neutral solutes that do not themselves directly affect the DNA–protein binding. This approach has been used to measure changes in hydration accompanying the DNA binding of several proteins: Escherichia coli gal repressor, E. coli CAP protein, Hin recombinase, Ultrabithorax and Deformed homeodomains, E. coli tyr repressor, EcoRI, and the Sso7d protein. Using an equilibrium competition approach, we showed previously that the free energy difference between complexes of the restriction nuclease EcoRI with nonspecific DNA and with the enzyme’s recognition sequence is linearly dependent on the change in water chemical potential of the solution with added osmolyte. This dependence translates into an additional ; 110 waters that are sequestered by the nonspecific complex relative to the specific complex at 20°C and ; 70 more waters at 4°C. This significant difference in retained waters between specific and nonspecific complexes is accompanied by a difference of ; 10 between the specific and nonspecific EcoRI DNA binding constants. The difference in hydration additionally was seen to be insensitive to the size and chemical nature of the solute used to change water activity for a wide variety of osmolytes. This result most probably implies that the water retained by the nonspecific complex is sequestered in a cavity at the DNA–protein interface that is sterically inaccessible to solutes.

Trapping DNA–protein binding reactions with neutral osmolytes for the analysis by gel mobility shift and self-cleavage assays

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.

Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV.

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.

Competition between netropsin and restriction nuclease EcoRI for DNA binding.

We find that netropsin and netropsin analogue protect DNA from EcorI restriction nuclease cleavage by inhibiting the binding of EcoRI to its recognition site. The drug -- EcoRI competitive binding constants measured by a electrophoretic gel mobility shift assay are in excellent agreement with the nuclease protection results for the netropsin analogue and in reasonable agreement for netropsin itself. Crystal structures of complexes show that netropsin and EcoRI recognize different regions of the DNA helix and would not be expected to compete for binding to the restriction nuclease site. The large distortions in DNA structure caused by EcoRI binding are most likely responsible for an indirect structural competition with netropsin binding. The structural change in the netropsin binding region induced by EcoRI binding to its region essentially prevents drug association. Given the reciprocal nature of competition, binding of netropsin to a minimally perturbed structure then also makes the association of EcoRI energetically more costly. Since many sequence specific DNA binding proteins significantly bend or distort the DNA helix, drugs that compete indirectly can be as effective as drugs that act through a direct steric inhibition.

Stabilizing labile DNA-protein complexes in polyacrylamide gels

The electrophoretic mobility‐shift assay (EMSA) is one of the most popular tools in molecular biology for measuring DNA–protein interactions. EMSA, as standardly practiced today, works well for complexes with association binding constants Ka>109 M−1 under normal conditions of salt and pH. Many DNA–protein complexes are not stable enough so that they dissociate while moving through the gel matrix giving smeared bands that are difficult to quantitate reliably. In this work we demonstrate that the addition of the osmolyte triethylene glycol to polyacrylamide gels dramatically stabilizes labile restriction endonuclease EcoRI complexes with nonspecific DNA sequences enabling quantitation of binding using EMSA. The significant improvement of the technique resulting from the addition of osmolytes to the gel matrix greatly extends the range of binding constants of protein–DNA complexes that can be investigated using this widely used assay. Extension of this approach to other techniques used for separating bound and free components such as gel chromatography and CE is straightforward.

Using single-turnover kinetics with osmotic stress to characterize the EcoRV cleavage reaction.

Type II restriction endonucleases require metal ions to specifically cleave DNA at canonical sites. Despite the wealth of structural and biochemical information, the number of Mg(2+) ions used for cleavage by EcoRV, in particular, at physiological divalent ion concentrations has not been established. In this work, we employ a single-turnover technique that uses osmotic stress to probe reaction kinetics between an initial specific EcoRV-DNA complex formed in the absence of Mg(2+) and the final cleavage step. With osmotic stress, complex dissociation before cleavage is minimized and the reaction rates are slowed to a convenient time scale of minutes to hours. We find that cleavage occurs by a two-step mechanism that can be characterized by two rate constants. The dependence of these rate constants on Mg(2+) concentration and osmotic pressure gives the number of Mg(2+) ions and water molecules coupled to each kinetic step of the EcoRV cleavage reaction. Each kinetic step is coupled to the binding 1.5-2.5 Mg(2+) ions, the uptake of ∼30 water molecules, and the cleavage of a DNA single strand. We suggest that each kinetic step reflects an independent, rate-limiting conformational change of each monomer of the dimeric enzyme that allows Mg(2+) ion binding. This modified single-turnover protocol has general applicability for metalloenzymes.

Stabilizing Labile DNA-Protein Complexes in Polyacrylamide Gels

The electrophoretic mobility shift assay (EMSA) is one of the most popular tools in molecular biology for measuring DNA-protein interactions. The technique uses polyacrylamide gel electrophoresis to separate DNA-protein or RNA-protein complexes from free DNA or RNA. Polyacrylamide gels stabilize DNA-protein, RNA-protein, or protein-protein complexes by a crowding or caging mechanism. Still every technique has its limitations. EMSA, as standardly practiced today, works well for complexes with association binding constants Ka>109 M−1 under normal conditions of salt and pH. Many DNA-protein complexes are not stable enough so that they dissociate while moving through the gel matrix giving smeared bands that are difficult to quantitate reliably. 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 stabilize complexes in the gel matrix as well as in the solution. In this work we demonstrate that the addition of the osmolyte triethylene glycol to polyacrylamide gels dramatically stabilizes labile restriction endonuclease EcoRI complexes with nonspecific DNA sequences enabling quantitation of binding using the electrophoretic mobility shift assay. The significant improvement of the technique resulting from the addition of osmolytes to the gel matrix greatly extends the range of binding constants of protein-DNA complexes that can be investigated using this widely used assay. Extension of this approach to other techniques used for separating bound and free components such as gel chromatography and capillary electrophoresis is straightforward.