EcoRI Binding Research Articles

Protein–DNA force assay in a microfluidic format

The detailed study of protein-DNA interactions is a core effort to elucidate physiological processes, including gene regulation, DNA repair and the immune response. The molecular force assay (MFA) is an established method to study DNA-binding proteins. In particular, high-affinity binder dissociation is made possible by the application of force. Microfluidic lab-on-a-chip approaches have proven helpful for parallelization, small sample volumes, reproducibility, and low cost. We report the successful combination of these two principles, forming a microfluidic molecular force assay and representing a novel use for the established MITOMI chip design. We present, characterize, validate and apply this integrated method. An alternative confocal fluorescence microscopy readout and analysis method is introduced and validated. In a multiplexing application, EcoRI binding is detected and characterized. This method paves the way for quantitative on-chip force measurements. It is suited for integration with DNA micro-spotting and in vitro expression of transcription factors to form a high-throughput chip for detailed DNA-protein interaction studies.

An FTIR Investigation of Flanking Sequence Effects on the Structure and Flexibility of DNA Binding Sites

Fourier transform infrared (FTIR) spectroscopy and a library of FTIR marker bands have been used to examine the structure and relative flexibilities conferred by different flanking sequences on the EcoRI binding site. This approach allowed us to examine unique peaks and subtle changes in the spectra of d(AAAGAATTCTTT)(2), d(TTCGAATTCGAA)(2), and d(CGCGAATTCGCG)(2) and thereby identify local changes in base pairing, base stacking, backbone conformation, glycosidic bond rotation, and sugar puckering in the studied sequences. The changes in flanking sequences induce differences in the sugar puckers, glycosidic bond rotation, and backbone conformations. Varying levels of local flexibility are observed within the sequences in agreement with previous biological activity assays. The results also provide supporting evidence for the presence of a splay in the G(4)-C(9) base pair of the EcoRI binding site and a potential pocket of flexibility at the G(4) cleavage site that have been proposed in the literature. In sum, we have demonstrated that FTIR is a powerful methodology for studying the effect of flanking sequences on DNA structure and flexibility, for it can provide information about the local structure of the nucleic acid and the overall relative flexibilities conferred by different flanking sequences.

31P NMR investigation of backbone dynamics in DNA binding sites.

The backbone conformation of DNA plays an important role in the indirect readout mechanisms for protein--DNA recognition events. Thus, investigating the backbone dynamics of each step in DNA binding sequences provides useful information necessary for the characterization of these interactions. Here, we use 31P dynamic NMR to characterize the backbone conformation and dynamics in the Dickerson dodecamer, a sequence containing the EcoRI binding site, and confirm solid-state 2H NMR results showing that the C3pG4 and C9pG10 steps experience unique dynamics and that these dynamics are quenched upon cytosine methylation. In addition, we show that cytosine methylation affects the conformation and dynamics of neighboring nucleotide steps, but this effect is localized to only near neighbors and base-pairing partners. Last, we have been able to characterize the percent BII in each backbone step and illustrate that the C3pG4 and C9pG10 favor the noncanonical BII conformation, even at low temperatures. Our results demonstrate that 31P dynamic NMR provides a robust and efficient method for characterizing the backbone dynamics in DNA. This allows simple, rapid determination of sequence-dependent dynamical information, providing a useful method for studying trends in protein-DNA recognition events.

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.

Probing Protein-DNA Interactions by Unzipping a Single DNA Double Helix

We present unzipping force analysis of protein association (UFAPA) as a novel and versatile method for detection of the position and dynamic nature of protein-DNA interactions. A single DNA double helix was unzipped in the presence of DNA-binding proteins using a feedback-enhanced optical trap. When the unzipping fork in a DNA reached a bound protein molecule we observed a dramatic increase in the tension in the DNA, followed by a sudden tension reduction. Analysis of the unzipping force throughout an unbinding “event” revealed information about the spatial location and dynamic nature of the protein-DNA complex. The capacity of UFAPA to spatially locate protein-DNA interactions is demonstrated by noncatalytic restriction mapping on a 4-kb DNA with three restriction enzymes ( BsoBI, XhoI, and EcoRI). A restriction map for a given restriction enzyme was generated with an accuracy of ∼25 bp. UFAPA also allows direct determination of the site-specific equilibrium association constant ( K A) for a DNA-binding protein. This capability is demonstrated by measuring the cation concentration dependence of K A for EcoRI binding. The measured values are in good agreement with previous measurements of K A over an intermediate range of cation concentration. These results demonstrate the potential utility of UFAPA for future studies of site-specific protein-DNA interactions.

Macromolecular Hydration Changes Associated with BamHI Binding and Catalysis

In this report, the effects of osmotic pressure on BamHI cognate binding and catalysis were investigated and compared with a previous study on EcoRI (Robinson, C. R. and Sligar, S. G. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 2186-2191). Our observation of the dependence of binding and catalytic parameters on osmotic pressure has allowed for the comparison of hydration changes associated with site-specific DNA recognition for both endonucleases. Over a large range of osmotic pressures (pi), the dependence of BamHI on osmotic stress during cognate binding and catalysis was very different from that of the related endonuclease EcoRI. The binding of EcoRI to cognate DNA was dominated by a dehydration of the endonuclease-DNA complex, whereas binding by BamHI to its cognate sequence was accompanied by a solvent release corresponding to some 125 fewer waters. Catalytic analysis at elevated osmotic pressures indicated that both endonucleases had undergone a net hydration of the complex with BamHI displaying a much greater dependence on osmotic stress than EcoRI. Although the enzymes shared core structural motifs, comparisons of high resolution x-ray structures revealed many different secondary structural features of the complexed endonucleases. The large difference in hydration changes by both BamHI and EcoRI could be attributed to these dissimilar secondary structural features, as well as the functional differences of the two endonucleases during site-specific DNA recognition.

Immobilization of the restriction endonuclease EcoRI usefulness of a polyclonal antibody support

The restriction enzyme EcoRI has been immobilized by using an immunoaffinity-based procedure on Sepharose 4B. The antibody support, prepared by coupling the γ-globulin fraction of serum of immunized rabbits to CNBr-activated Sepharose-4B, was highly effective in binding EcoRI from solution although only about half of the bound activity appeared to be expressed by the immobilized preparations. Restriction activity of EcoRI immobilized on the antibody support was indistinguishable from that of soluble enzyme on the linear λ-DNA and supercoiled plasmids pBR322 and pBHLUC P. Binding to the antibody support markedly enhanced the resistance of EcoRI to heat inactivation, and the preparation, unlike the native enzyme, retained significant activity after exposure to a temperature of 65°C. It was also possible to immobilize EcoRI directly from the cell lysates of Escherichia coli pMB4, an EcoRI overproducing strain. The immobilized preparation did not possess nonspecific nuclease activity that was prominent in the lysates, suggesting specificity in the binding of EcoRI to the antibody support.

Changes in solvation during DNA binding and cleavage are critical to altered specificity of the EcoRI endonuclease.

Restriction endonucleases such as EcoRI bind and cleave DNA with great specificity and represent a paradigm for protein-DNA interactions and molecular recognition. Using osmotic pressure to induce water release, we demonstrate the participation of bound waters in the sequence discrimination of substrate DNA by EcoRI. Changes in solvation can play a critical role in directing sequence-specific DNA binding by EcoRI and are also crucial in assisting site discrimination during catalysis. By measuring the volume change for complex formation, we show that at the cognate sequence (GAATTC) EcoRI binding releases about 70 fewer water molecules than binding at an alternate DNA sequence (TAATTC), which differs by a single base pair. EcoRI complexation with nonspecific DNA releases substantially less water than either of these specific complexes. In cognate substrates (GAATTC) kcat decreases as osmotic pressure is increased, indicating the binding of about 30 water molecules accompanies the cleavage reaction. For the alternate substrate (TAATTC), release of about 40 water molecules accompanies the reaction, indicated by a dramatic acceleration of the rate when osmotic pressure is raised. These large differences in solvation effects demonstrate that water molecules can be key players in the molecular recognition process during both association and catalytic phases of the EcoRI reaction, acting to change the specificity of the enzyme. For both the protein-DNA complex and the transition state, there may be substantial conformational differences between cognate and alternate sites, accompanied by significant alterations in hydration and solvent accessibility.

Differences in water release for the binding of EcoRI to specific and nonspecific DNA sequences.

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 water chemical potential of the solution, set using several very different solutes, ranging from glycine and glycerol to triethylene glycol and sucrose. This osmotic dependence indicates that the nonspecific complex sequesters some 110 waters more than the specific complex with the recognition sequence. The insensitivity of the difference in number of waters released to the solute identity further indicates that this water is sequestered in a space that is sterically inaccessible to solutes, most likely at the protein-DNA interface of the nonspecific complex. Calculations based on the structure of the specific complex suggest that the apposing DNA and protein surfaces in the nonspecific complex retain approximately a full hydration layer of water.

Direct atomic force microscope imaging of EcoRI endonuclease site specifically bound to plasmid DNA molecules.

Direct imaging with the atomic force microscope has been used to identify specific nucleotide sequences in plasmid DNA molecules. This was accomplished using EcoRI (Gln-111), a mutant of the restriction enzyme that has a 1000-fold greater binding affinity than the wild-type enzyme but with cleavage rate constants reduced by a factor of 10(4). ScaI-linearized plasmids with single (pBS+) and double (pGEM-luc and pSV-beta-galactosidase) EcoRI recognition sites were imaged, and the bound enzyme was localized to a 50- to 100-nt resolution. The high affinity for the EcoRI binding site exhibited by this mutant endonuclease, coupled with an observed low level of nonspecific binding, should prove valuable for physically mapping large DNA clones by direct atomic force microscope imaging.

Hybridization of fluorescein-labeled DNA oligomers detected by fluorescence anisotropy with protein binding enhancement.

Fundamental aspects of the application of fluorescence anisotropy to detect the hybridization of fluorescein-labeled DNA oligomers were explored. The oligomers included a binding site for the EcoRI restriction enzyme, which binds to double-stranded DNA and is used in this work to enhance the difference between the anisotropies of the single-stranded and double-stranded oligomers by increasing the effective volume of the latter. The fluorescence anisotropy increases upon hybridization and further upon binding of EcoRI to the double strand. By varying the length of the tether used to attach the fluorescein to the 5' end of the oligonucleotide, it was found that a 6-carbon tether was optimal, providing the most dramatic increases in anisotropy in the presence of EcoRI. Dynamic fluorescence anisotropy (DFA) provided insight into the increases in steady-state anisotropy. In most cases, the best fits to the DFA data were obtained using a biexponential decay model, which describes an anisotropic rotator. Upon hybridization, the faster rotational motion is more hindered, and the contribution of the slower rotational component is increased. This effect is enhanced by binding of EcoRI to the double strand, especially when the EcoRI binding site is near the fluorescein at the 5' end and the tether length is in the optimal range. Because the rotational correlation time of the slower anisotropy decay component is much longer than the fluorescence lifetime, it is possible in some cases to reduce the anisotropic rotator model to the special limiting case of a hindered rotator.

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.

A Solid-State 2H NMR Investigation of Purine Motion in a 12-Base-Pair RNA Duplex

Solid-state 2H NMR spectroscopy has been used to investigate the base dynamics of a RNA oligonucleotide with a defined sequence, [r(CGCGAAUUCGCG)] 2, which contains the RNA analogue of the EcoRI binding site. The C8 protons of all purines in the self-complementary dodecamer were exchanged for deuterons. The quadrupole-echo lineshapes and spin-lattice relaxation times as a function of hydration for the sample in the form of the Na salt have previously been reported. In that study the 2H NMR lineshapes and T 1 values of [r(CG*CG*A*A*UUCG*CG*)] 2 were compared with those of the analogously labeled DNA sequence, [d(CG*CG*A*A*TTCG*CG*)](2) (Wang et al., J.Am. Chem. Soc. 114, 6583, 1992). It was concluded that the amplitudes of purine motion for DNA and RNA are similar at all hydration levels; however, the rate difference observed at low-hydration levels may or may not persist at high hydration. Here the internal motions of the purine bases in the RNA oligomer have been thoroughly investigated. Three models were used to simulate the motion: (1) two-site jump, (2) diffusion in a cone, and (3) restricted diffusion on the surface of a cone. The purine motion is best simulated by the restricted-diffusion on a cone model with an amplitude of ±9.5° and a rate between 8.0 × 10 6 rad/s at 90% RH and 8.4 × 10 8 rad/s at 0% RH. This small amplitude and fast rate of purine motion for RNA are similar to previous results obtained for DNA purines.

Dynamics of bases in hydrated [d(CGCGAATTCGCG)]2.

Solid-state 2H NMR spectroscopy has been used to investigate the dynamics of a DNA oligonucleotide with a defined sequence, [d(CGCGAATTCGCG)]2, which contains the EcoRI binding site. Quadrupole echo line shapes and spin-lattice relaxation times were obtained as a function of hydration on two different deuterated samples, both in the form of the Na salt. In one sample, the C8 protons of all purines in the self-complementary dodecamer were exchanged for deuterons. In the other sample, a specifically labeled thymidine (C6 deuterated) was synthetically incorporated at the seventh position (counting 5' to 3') in the sequence. The general trends for both samples were quite similar. At all levels of hydration, the data reveal the presence of a rapid, small-amplitude libration of the bases (tau c less than or equal to 1 ns, 6 degrees-10 degrees amplitude). At the higher hydration levels (80% relative humidity or higher), the results indicate the presence of a much slower motion (tau c approximately 10-100 microseconds), which at 80% relative humidity is of small amplitude (approximately 5 degrees) and at higher hydration levels may be of larger amplitude. There is no evidence for large-amplitude (greater than +/- 10 degrees) motion on a nanosecond or faster time scale under any hydration condition. The 2H NMR results were analyzed with a dynamical model which treats the oligonucleotide as a deformable filament and which can include collective torsional fluctuations. The slow motion observed at high hydration levels is attributed to the uniform twisting mode (of the entire helix).(ABSTRACT TRUNCATED AT 250 WORDS)

Restriction endonuclease EcoRI alters the enantiomeric preference of chiral metallointercalators for DNA: an illustration of a protein-induced DNA conformational change.

A conformational change in the DNA plasmid ColE1 appears to occur upon specific binding of the restriction endonuclease EcoRI. Enzyme association alters the chiral discrimination found in binding metallointercalators to DNA sites. The complexes tris(1,10-phenanthroline)ruthenium(II), Ru(phen)3(2+), tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II), Ru(DIP)3(2+), and tris(4,7-diphenyl-1,10-phenanthroline)cobalt(III), Co(DIP)3(3+), in general, bind stereoselectively to DNA helices, with enantiomers possessing the delta configuration bound preferentially by right-handed B-DNA. In the presence of EcoRI, however, this enantioselectivity is altered. The chiral intercalators, at micromolar concentrations, inhibit the reaction of EcoRI, but for each enantiomeric pair it is the lambda enantiomer, which binds only poorly to a B-DNA helix, that inhibits EcoRI preferentially. Kinetic studies in the presence of lambda-Ru(DIP)3(2+) indicate that the enzyme inhibition occurs as a result of the lambda enantiomer binding to the enzyme-DNA complex as well as to the free enzyme. Furthermore, photolytic strand cleavage experiments using Co(DIP)3(3+) indicate that the metal complex interacts directly at the protein-bound DNA site. Increasing concentrations of bound EcoRI stimulate photoactivated cleavage of the DNA helix by lambda-Co(DIP)3(3+), until a protein concentration is reached where specific DNA recognition sites are saturated with enzyme. Thus, although lambda-Co(DIP)3(3+) does not bind closely to the DNA in the absence of enzyme, specific binding of EcoRI appears to alter the DNA structure so as to permit the close association of the lambda isomer to the DNA helix. Mapping experiments demonstrate that this association leads to photocleavage of DNA by the cobalt complex at or very close to the EcoRI recognition site.(ABSTRACT TRUNCATED AT 250 WORDS)