Photogeneration of hydroxyl radicals for footprinting.

Hydroxyl radicals yield footprints of DNA-ligand interactions that are uniform in intensity and display single base pair resolution. It is shown here that brief illumination of dilute aqueous solutions of hydrogen peroxide with a standard uv transilluminator can be used to generate hydroxyl radicals for footprinting studies. Photogenerated hydroxyl radicals are used to footprint netropsin, a drug that interacts with the minor groove of DNA. The method presented eliminates two of the reagents used in conventional Fenton-reaction-based hydroxyl radical footprinting. It has the further advantage that the extent of cleavage of the DNA can be precisely regulated by controlling the illumination time. Because light is used to drive the reaction, photogenerated hydroxyl radicals can be used to footprint DNA-ligand interactions under experimental conditions of temperature and pressure inaccessible to Fenton-reaction chemistry.     

High resolution footprinting of EcoRI and distamycin with Rh(phi)2(bpy)3+, a new photofootprinting reagent.

The complex bis(phenanthrenequinone diimine)(bipyridyl)rhodium(III), Rh(phi)2(bpy)3+, cleaves DNA efficiently in a sequence-neutral fashion upon photoactivation so as to provide a novel, high resolution, chemical photofootpring reagent. Photofootprinting of two crystallographically characterized DNA-binding agents, distamycin, a small natural product which binds to DNA in the minor groove, and the endonuclease EcoRI, which binds in the major groove, gave respectively a 5-7 base pair footprint for the drug at its A6 binding site and a 10-12 base pair footprint for the enzyme centered at its recognition site (5'-GAATTC-3'). Both footprints agree closely with the crystallographic results. The photocleavage reaction can be performed using either a high intensity lamp or, conveniently, a simple transilluminator box, and the photoreaction is not inhibited by moderate concentrations of reagents which are sometimes required for examining interactions of molecules with DNA. When compared with other popular footprinting agents, the rhodium complex shows a number of distinct advantages: sequence-neutrality, high resolution, ability to footprint major as well as minor groove-binding ligands, applicability in the presence of additives such as Mg2+ or glycerol, ease of handling, and a sharply footprinted pattern. Light activated footprinting reactions furthermore offer the possibility of examining DNA-binding interactions with time resolution and within the cell.     

Removing water from an EcoRI-noncognate DNA complex with osmotic stress.

We recently showed that a nonspecific complex of the restriction nuclease EcoRI with poly (dI-dC) sequesters significantly more water at the protein-DNA interface than the complex with the specific recognition sequence. The nonspecific complex seems to retain almost a full hydration layer at the interface. We now find that at low osmotic pressures a complex of the restriction nuclease EcoRI with a DNA sequence that differs by only one base pair from the recognition site (a 'star' sequence) sequesters about 70 waters more than the specific one, a value virtually indistinguishable from nonspecific DNA. Unlike complexes with oligo (dI-dC) or with a sequence that differs by two base pairs from the recognition sequence, however, much of the water in the 'star' sequence complex is removed at high osmotic pressures. The energy of removing this water can be calculated simply from the osmotic pressure work done on the complex. The ability to measure not only the changes in water sequestered by DNA-protein complexes for different sequences, but also the work necessary to remove this water is a potentially powerful new tool for coupling inferred structural changes and thermodynamics.     

Base pair opening within B-DNA: free energy pathways for GC and AT pairs from umbrella sampling simulations

The conformational pathways and the free energy variations for base opening into the major and minor grooves of a B-DNA duplex are studied using umbrella sampling molecular dynamics simulations. We compare both GC and AT base pair opening within a double-stranded d(GAGAGAGAGAGAG)· d(CTCTCTCTCTCTC) oligomer, and we are also able to study the impact of opening on the conformational and dynamic properties of DNA and on the surrounding solvent. The results indicate a two-stage opening process with an initial coupling of the movements of the bases within the perturbed base pair. Major and minor groove pathways are energetically comparable in the case of the pyrimidine bases, but the major groove pathway is favored for the larger purine bases. Base opening is coupled to changes in specific backbone dihedrals and certain helical distortions, including untwisting and bending, although all these effects are dependent on the particular base involved. Partial opening also leads to well defined water bridging sites, which may play a role in stabilizing the perturbed base pairs.     

Thermodynamic and Structural Basis for Relaxation of Specificity in Protein–DNA Recognition

As a novel approach to the structural and functional properties that give rise to extremely stringent sequence specificity in protein–DNA interactions, we have exploited “promiscuous” mutants of EcoRI endonuclease to study the detailed mechanism by which changes in a protein can relax specificity. The A138T promiscuous mutant protein binds more tightly to the cognate GAATTC site than does wild-type EcoRI yet displays relaxed specificity deriving from tighter binding and faster cleavage at EcoRI* sites (one incorrect base pair). AAATTC EcoRI* sites are cleaved by A138T up to 170-fold faster than by wild-type enzyme if the site is abutted by a 5′-purine-pyrimidine (5′-RY) motif. When wild-type protein binds to an EcoRI* site, it forms structurally adapted complexes with thermodynamic parameters of binding that differ markedly from those of specific complexes. By contrast, we show that A138T complexes with 5′-RY-flanked AAATTC sites are virtually indistinguishable from wild-type-specific complexes with respect to the heat capacity change upon binding (?C°_P), the change in excluded macromolecular volume upon association, and contacts to the phosphate backbone. While the preference for the 5′-RY motif implicates contacts to flanking bases as important for relaxed specificity, local effects are not sufficient to explain the large differences in ?C°_P and excluded volume, as these parameters report on global features of the complex. Our findings therefore support the view that specificity does not derive from the additive effects of individual interactions but rather from a set of cooperative events that are uniquely associated with specific recognition.     

Crystal structure of MunI restriction endonuclease in complex with cognate DNA at 1.7 A resolution.

The MunI restriction enzyme recognizes the palindromic hexanucleotide sequence C/AATTG (the '/' indicates the cleavage site). The crystal structure of its active site mutant D83A bound to cognate DNA has been determined at 1.7 A resolution. Base-specific contacts between MunI and DNA occur exclusively in the major groove. While DNA-binding sites of most other restriction enzymes are comprised of discontinuous sequence segments, MunI combines all residues involved in the base-specific contacts within one short stretch (residues R115-R121) located at the N-terminal region of the 3(10)4 helix. The outer CG base pair of the recognition sequence is recognized solely by R115 through hydrogen bonds made by backbone and side chain atoms to both bases. The mechanism of recognition of the central AATT nucleotides by MunI is similar to that of EcoRI, which recognizes the G/AATTC sequence. The local conformation of AATT deviates from the typical B-DNA form and is remarkably similar to EcoRI-DNA. It appears to be essential for specific hydrogen bonding and recognition by MunI and EcoRI.     

The Opening of a Single Base Without Perturbations of Neighboring Nucleotides: A Study on Crystal B-DNA duplex d(CGCGAATTCGCG)2

Abstract

In this work we explore the possibility of the opening of a single base without perturbation of its neighboring nucleotides. Low energy base opening into the grooves can be accomplished by rotation of the relevant backbone and glycosidic bond torsion angles. The pathway has been determined by identifying ζ torsion angle as the reaction coordinate together with the accompanying geometric requirement that guides the displacement of other torsion angles. Our study on Dickerson dodecamer duplex d(CGCGAATTCGCG)₂ showed that all bases with normal equilibrium ζ can be rotated by ~ 30°, corresponding to ~ 3.5Å base displacement, towards the major groove. Such an opening extent is comparable with estimated amplitudes of local angular motions in DNA bases from NMR experiments, which might facilitate proton exchange. The computed base opening energy barrier is also comparable with measured base pair opening enthalpy. These results indicate possible relevance of the pathway studied in this work with experimentally observed base pair opening process. Our analysis also showed a preference for base opening along the major groove and an abnormal behavior for bases with unusual equilibrium ζ torsion angle.     

Variable Temperature Infrared Spectroscopy of Cytosine-Guanine Base Pairs: Tautomerism Versus Polarization

Abstract

This report describes an infrared (IR) spectroscopic study of a model cytosine - guanine base pair. This base pair is part of a self-consistent experimental system based on lipophilic ribose derivatives of cytidine (C), guanosine (G) and O⁶-methylguanosine (O⁶MeG) that are soluble in non-aqueous, low dielectric solvents at appreciable concentrations. Previous experiments on this system have revealed different rotation dynamics for the amino bonds within the CG base pair, an observation that could be explained by the presence of rare tautomers (P.O. Lowdin, Reviews of Modern Physics 35, 724 (1963)), or by mutual polarization of the base pairs (L.D. Williams, N.G. Williams and B.R. Shaw, J. Am. Chem. Soc. 112, 829 (1990)). The IR spectra in the OH and NH stretching region indicate formation of hydrogen-bonded CG base pairs and self associates in 1,2-dichlorobenzene over a temperature range from 10 to 290K. Changes in the lineshapes and intensities of the IR bands with temperature correlate with phase transitions of the solvent but no evidence is seen for an OH stretching band that would indicate the formation of hydroxyl tautomers within base pairs. Similarly, the relative intensities of the C=O stretching bands of CG in cyclohexane solution remain constant over this same temperature range, confirming that within the base pair, the tautomeric states of the bases remain essentially unperturbed in the 2-amino/6-keto form of G and the 2-keto/4-amino form of C. The spectra of O⁶-MeG aid in the band assignments, since this molecule is frozen in an equivalent of the 2-amino/6-hydroxyl tautomer, but without the OH group and its associated stretching band. We conclude that the probability of tautomerism does not appear to be sufficient to explain the different rotation dynamics for the two amino bonds of the CG base pair. Rather it is argued that mutual polarization within the base pair, which would increase the bond order of the amino bond of C within the base pair, can explain the results without the formation of unconventional tautomers.     

Variable temperature infrared spectroscopy of cytosine-guanine base pairs: tautomerism versus polarization.

This report describes an infrared (IR) spectroscopic study of a model cytosine-guanine base pair. This base pair is part of a self-consistent experimental system based on lipophilic ribose derivatives of cytidine (C), guanosine (G) and O6-methylguanosine (O6MeG) that are soluble in non-aqueous, low dielectric solvents at appreciable concentrations. Previous experiments on this system have revealed different rotation dynamics for the amino bonds within the CG base pair, an observation that could be explained by the presence of rare tautomers (P.O. Lowdin, Reviews of Modern Physics 35,724 (1963)), or by mutual polarization of the base pairs (L.D. Williams, N.G. Williams and B.R. Shaw,J.Am.Chem.Soc. 112,829 (1990)). The IR spectra in the OH and NH stretching region indicate formation of hydrogen-bonded CG base pairs and self associates in 1,2-dichlorobenzene over a temperature range from 10 to 290K. Changes in the lineshapes and intensities of the IR bands with temperature correlate with phase transitions of the solvent, but no evidence is seen for an OH stretching band that would indicate the formation of hydroxyl tautomers within base pairs. Similarly, the relative intensities of the C = O stretching bands of CG in cyclohexane solution remain constant over this same temperature range, confirming that within the base pair, the tautomeric states of the bases remain essentially unperturbed in the 2-amino/6-keto form of G and the 2-keto/4-amino form of C. The spectra of O6-MeG aid in the band assignments, since this molecule is frozen in an equivalent of the 2-amino/6-hydroxyl tautomer, but without the OH group and its associated stretching band. We conclude that the probability of tautomerism does not appear to be sufficient to explain the different rotation dynamics for the two amino bonds of the CG base pair. Rather it is argued that mutual polarization within the base pair, which would increase the bond order of the amino bond of C within the base pair, can explain the results without the formation of unconventional tautomers.     

Linear free energy correlations for enzymatic base flipping: how do damaged base pairs facilitate specific recognition?

To efficiently maintain their genomic integrity, DNA repair glycosylases must exhibit high catalytic specificity for their cognate damaged bases using an extrahelical recognition mechanism. One possible contribution to specificity is the weak base pairing and inherent instability of damaged sites which may lead to increased extrahelicity of the damaged base and enhanced recognition of these sites. This model predicts that the binding affinity of the enzyme should increase as the thermodynamic stability of the lesion base pair decreases, because less work is required to extrude the base into its active site. We have tested this hypothesis with uracil DNA glycosylase (UDG) by constructing a series of DNA duplexes containing a single uracil (U) opposite a variety of bases (X) that formed from zero to three hydrogen bonds with U. Linear free energy (LFE) relationships were observed that correlated UDG binding affinity with the entropy and enthalpy of duplex melting, and the dynamic accessibility of the damaged site to chemical oxidation. These LFEs indicate that the increased conformational freedom of the damaged site brought about by enthalpic destabilization of the base pair promotes the formation of extrahelical states that enhance specific recognition by as much as 3000-fold. However, given the small stability differences between normal base pairs and U.A or U.G base pairs, relative base pair stability contributes little to the >10(6)-fold discrimination of UDG for uracil sites in cellular DNA. In contrast, the intrinsic instability of other more egregious DNA lesions may contribute significantly to the specificity of other DNA repair enzymes that bind to extrahelical bases.     

Isolation, cloning and characterization of argF gene DNA from Escherichia coli K-12.

A 1650 base pair (BP) fragment carrying the entire argF structural gene with its associated control regions was isolated from an EcoRI/BamHI digest of phi80argFilambda cI857 DNA. This segment was cloned using the EcoRI and BamHI cleavage sites in the plasmid pBR322. A preliminary restriction map of the argF region was prepared. RNA polymerase binding studies indicated that the argF promoter is located approx. 30 base pairs from the EcoRI terminus of the cloned DNA segment.     

Thermodynamic properties of the specific binding between Ag+ ions and C:C mismatched base pairs in duplex DNA

Metal-mediated base pairs formed by the interaction between metal ions and artificial bases in oligonucleotides have been developed for potential applications in nanotechnology. We recently found that a natural C:C mismatched base pair bound to an Ag(+) ion to generate a novel metal-mediated base pair in duplex DNA. Preparation of the novel C-Ag-C base pair involving natural bases is more convenient than that of metal-mediated base pairs involving artificial bases because time-consuming base synthesis is not required. Here, we examined the thermodynamic properties of the binding between the Ag(+) ion and each of single and double C:C mismatched base pair in duplex DNA by isothermal titration calorimetry. The Ag(+) ion specifically bound to the C:C mismatched base pair at a 1:1 molar ratio with 10(6) M(-1) binding constant, which was significantly larger than those for nonspecific metal ion-DNA interactions. The specific binding between the Ag(+) ion and the single C:C mismatched base pair was mainly driven by the positive dehydration entropy change and the negative binding enthalpy change. In the interaction between the Ag(+) ion and each of the consecutive and interrupted double C:C mismatched base pairs, stoichiometric binding at a 1:1 molar ratio was achieved in each step of the first and second Ag(+) binding. The binding affinity for the second Ag(+) binding was similar to that for the first Ag(+) binding. Stoichiometric binding without interference and negative cooperativity may be favorable for aligning multiple Ag(+) ions in duplex DNA for applications of the metal-mediated base pairs in nanotechnology.     

Structures of Oligonucleotides Containing 5-(methoxymethyl)-2′-deoxyuridine Determined by NMR Spectroscopy

Abstract

Base pairing of 5-(methoxymethyl)-2′-deoxyuridine (MMdU) opposite either adenine or guanine in a seven base pair oligonucleotide duplex has been studied by NMR spectroscopy. When paired with A, we observe that the MMdU. Abase pair adopts Watson-Crick geometry. The methoxymethyl substituent is not held in a fixed conformation and may rotate around the C5-CH₂ and CH₂?O bonds. Examination of the potential energy as a function of rotation around these bonds indicates the presence of four low energy conformations. No hydrogen bonding is indicated for the methoxymethyl substituent, and the four potential minima result from reduced steric clash. For the MMdU. G base pair, the two bases adopt a wobble geometry which does not change with increasing solvent pH. Similarly, we find four low energy conformations for the methoxymethyl substituent in the major groove of the DNA helix.     

Effects of functional group changes in the EcoRI recognition site on the cleavage reaction catalyzed by the endonuclease

Oligodeoxynucleotides have been prepared that contain changes in the functional group pattern present in the EcoRI recognition site. These changes involve "functional group deletions", "functional group reversals", and "displaced functional groups". Steady-state kinetic parameters have been used to characterize the interaction of these modified recognition sites with the EcoRI endonuclease. Changes in the functional group pattern have varying effects upon the cleavage reaction. Both the exocyclic amino groups of the two adenine residues and the methyl groups of the thymine residues appear to interact with the endonuclease quite differently. In both cases efficient catalysis was observed when these functional groups were present at the "outer" dA-dT base pair. Selectivity was decreased by over an order of magnitude largely via increases in Km when these functional groups were deleted. Similar modifications at the "inner" dA-dT base pair did not alter the kinetic parameters significantly from those observed with the native sequence. Addition of an amino group to the minor groove at the outer dA-dT base pair resulted in a modified recognition site that interacted with the enzyme, on the basis of observed competitive inhibition kinetics, but was not cleaved.     

Kinds of mutations formed when a shuttle vector containing adducts of benzo[a]pyrene-7,8-diol-9,10-epoxide replicates in COS7 cells.

We have investigated the kinds of mutations induced when a shuttle vector containing covalently bound residues of the (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) replicates in the monkey kidney cell line COS7. The target for detecting mutations was the 200-base pair gene for a tyrosine suppressor tRNA (supF), inserted at the EcoRI site in shuttle vector p3AC (Sarkar et al., Mol. Cell. Biol. 4:2227-2230, 1984). When introduced by transformation, a functioning supF gene in progeny plasmid recovered from COS7 cells allows suppression of a lacZ amber mutation in the indicator Escherichia coli host. Treatment of p3AC with BPDE caused a linear increase in the number of BPDE residues bound per plasmid. Untreated plasmids and plasmids containing 6.6 BPDE residues were transfected into COS7 cells, and the progeny were assayed for mutations in the supF gene. The frequency of mutants generated during replication of the BPDE-treated plasmids was not higher than that from untreated plasmids, but the two populations differed markedly in the kinds of mutations they contained. Gel electrophoresis analysis of the size alterations of 77 mutant plasmids obtained with untreated DNA and 45 obtained with BPDE-treated DNA showed that the majority of the mutant progeny of untreated plasmids exhibited gross alterations, principally large deletions. In contrast, the majority of the mutants generated during replication of the BPDE-treated plasmids contained only minor alterations, principally point mutations. Sequence analysis of progeny of untreated plasmids containing putative point mutations showed insertions and deletions of bases and a broad spectrum of base substitutions; in those from BPDE-treated plasmids, all base substitutions involved guanosine . cystosine pairs.     

Conformational properties of the G.G mismatch in d(CGCGAATTGGCG)2 determined by NMR.

The conformational properties of the DNA duplex d(CGCGAATTGGCG)2, which contains two noncomplementary G.G base pairs, have been examined in aqueous solution by 1H and 31P NMR as a function of temperature. The G.G mismatch is highly destabilizing, with a Tm value 35 K below that observed for the native EcoRI dodecamer. The dodecamer appears symmetric in the NMR spectra and exists largely as an average B-type DNA conformation. However, the 1H and 31P NMR spectra give evidence of considerable conformational heterogeneity at the mismatched nucleotides and their nearest neighbors, which increases with increasing temperature. There is no evidence for a significant population of the syn purine conformation. The imino protons of the mispaired bases G4 and G9 are degenerate, resonate at high field, and exchange readily with solvent. These results indicate that the mispaired bases are only weakly hydrogen-bonded and are only partially stacked into the helix. On raising the temperature, the duplex shows increasing exchange between two or more conformations originating from the mismatch sites. However, these additional conformations maintain their Watson-Crick hydrogen bonding. The increase in chemical exchange is consistent with a quasimelting process for which the G.G sites provide local nuclei. Extensive modeling studies by dynamic annealing have confirmed that the G(anti).G(anti) conformation is favored and that the mispairs are poorly stacked within the helix. The results explain both the poor thermal stability and low hypochromicity of this duplex.     

Pressure effect on the conformational fluctuation of apomyoglobin in the native state

We have investigated the effect of pressure on fluctuations of the native state of sperm whale apomyoglobin (apoMb) by H/D exchange, fluorescence, and limited proteolysis. The results from intrinsic fluorescence showed that a large fraction of apoMb molecules is in the native conformation in the pressure range from 0.1 to 150 MPa at 293 K and pH 6.0. The H/D exchange of protons of the individual backbone amino acids in this pressure range was monitored by NMR. The rate of H/D exchange was enhanced at high pressure, with the protection factors for some residues decreasing by factors of more than 100 compared to the values at 0.1 MPa. The amplitude of the decrease of the protection factor varied among the individual amino acids on the same secondary structure unit. This result suggests that H/D exchange in apoMb is explained best by the penetration model, in which solvent penetrates into the protein matrix via small motions. The result from limited proteolysis under high pressure showed that a pressure increase does not induce local unfolding of the secondary structure units of apoMb. Conformational fluctuations much smaller than local unfolding evidently provide pathways for water to diffuse into the protein interior, and are enhanced by an increase of pressure.     

An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules

Toward the expansion of the genetic alphabet, we present an unnatural base pair system for efficient PCR amplification, enabling the site-specific incorporation of extra functional components into DNA. This system can be applied to conventional PCR protocols employing DNA templates containing unnatural bases, natural and unnatural base triphosphates, and a 3′→5′ exonuclease-proficient DNA polymerase. For highly faithful and efficient PCR amplification involving the unnatural base pairing, we identified the natural-base sequences surrounding the unnatural bases in DNA templates by an in vitro selection technique, using a DNA library containing the unnatural base. The system facilitates the site-specific incorporation of a variety of modified unnatural bases, linked with functional groups of interest, into amplified DNA. DNA fragments (0.15 amol) containing the unnatural base pair can be amplified 10⁷-fold by 30 cycles of PCR, with <1% total mutation rate of the unnatural base pair site. Using the system, we demonstrated efficient PCR amplification and functionalization of DNA fragments for the extremely sensitive detection of zeptomol-scale target DNA molecules from mixtures with excess amounts (pmol scale) of foreign DNA species. This unnatural base pair system will be applicable to a wide range of DNA/RNA-based technologies.