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Jacqueline K Barton

Publications and source records attributed to Jacqueline K Barton.

At least 19 recordsLinked to original sources
PubMed2007

Differential ionic permeation of DNA-modified electrodes.

Ionic permselectivity of DNA films has been investigated by the analysis of the electrochemical response of methylene blue (MB) as a function of pH and ionic strength on DNA-modified electrodes in aqueous p-nitrophenol (p-NP) and phosphate buffers. We have observed a linear Pourbaix diagram in p-NP buffer indicating that the reduction of MB occurs with a two-electron plus one-proton reaction. Interestingly, in phosphate buffer the Pourbaix diagram is curved and this suggests that the thermodynamics of MB incorporated in the film depend also on the ratio of mono- versus divalent anions in the bulk. This result indicates that DNA films do not behave as pure ion-exclusion films, but instead there is a differential permselectivity that depends on the identity of the anions. Based on this consideration of the ionic distribution in the films, we provide a new method for the analysis of the DNA surface coverage based on AC impedance of an anionic species, ferricyanide. The methodology is of particular value in analyzing DNA hybridization and dehybridization. This approach presents an advantage compared to standard ruthenium hexammine assays since our methodology is insensitive to film morphology, and is highly sensitive to the amount of negative charge on the surface.

Buffers↗
PubMed2006

Insights into finding a mismatch through the structure of a mispaired DNA bound by a rhodium intercalator.

We report the 1.1-A resolution crystal structure of a bulky rhodium complex bound to two different DNA sites, mismatched and matched in the oligonucleotide 5'-(dCGGAAATTCCCG)2-3'. At the AC mismatch site, the structure reveals ligand insertion from the minor groove with ejection of both mismatched bases and elucidates how destabilized mispairs in DNA may be recognized. This unique binding mode contrasts with major groove intercalation, observed at a matched site, where doubling of the base pair rise accommodates stacking of the intercalator. Mass spectral analysis reveals different photocleavage products associated with the two binding modes in the crystal, with only products characteristic of mismatch binding in solution. This structure, illustrating two clearly distinct binding modes for a molecule with DNA, provides a rationale for the interrogation and detection of mismatches.

Base Pair Mismatch↗
PubMed2006

Targeting DNA mismatches with rhodium intercalators functionalized with a cell-penetrating peptide.

Cell-penetrating peptides are widely used to deliver cargo molecules into cells. Here we describe the synthesis, characterization, DNA binding, and cellular uptake studies of a series of metal-peptide conjugates containing oligoarginine as a cell-penetrating peptide. d-Octaarginine units are appended onto a rhodium intercalator containing the sterically expansive chrysenequinone diimine (chrysi) ligand to form Rh(chrysi)(phen)(bpy)(3+)-tethered oligoarginine conjugates, where the peptide is attached to the ancillary bpy ligand; some conjugates also include a fluorescein or thiazole orange tag. These complexes bind and with photoactivation selectively cleave DNA neighboring single-base mismatches. The presence of the oligoarginines is found to increase the nonspecific binding affinity of the complexes for both matched and mismatched DNA, but for these conjugates, photocleavage remains selective for the mismatched site, as assayed using both gel electrophoresis and mass spectrometry experiments. Significantly, the rhodium complex does not interfere with the delivery properties of the cell-penetrating peptide. Confocal microscopy experiments show rapid nuclear localization of the metal-peptide conjugates containing the tethered fluorescein. Mass spectrometry experiments confirm the association of the rhodium with the HeLa cells. These results provide a strategy for targeting mismatch-selective metal complexes inside cell nuclei.

Arginine↗
PubMed2006

DNA mismatch-specific targeting and hypersensitivity of mismatch-repair-deficient cells to bulky rhodium(III) intercalators.

Mismatch repair (MMR) is critical to maintaining the integrity of the genome, and deficiencies in MMR are correlated with cancerous transformations. Bulky rhodium intercalators target DNA base mismatches with high specificity. Here we describe the application of bulky rhodium intercalators to inhibit cellular proliferation differentially in MMR-deficient cells compared with cells that are MMR-proficient. Preferential inhibition by the rhodium complexes associated with MMR deficiency is seen both in a human colon cancer cell line and in normal mouse fibroblast cells; the inhibition of cellular proliferation depends strictly on the MMR deficiency of the cell. Furthermore, our assay of cellular proliferation is found to correlate with DNA mismatch targeting by the bulky metallointercalators. It is the Delta-isomer that is active both in targeting base mismatches and in inhibiting DNA synthesis. Additionally, the rhodium intercalators promote strand cleavage at the mismatch site with photoactivation, and we observe that the cellular response is enhanced with photoactivation. Targeting DNA mismatches may therefore provide a cell-selective strategy for chemotherapeutic design.

Animals↗
PubMed2006

Direct electrochemistry of endonuclease III in the presence and absence of DNA.

The electrochemistry of the base excision repair enzyme Endonuclease III (Endo III) in the presence and absence of DNA has been examined on highly oriented pyrolytic graphite (HOPG). At the surface modified with pyrenated DNA, a reversible signal is observed at 20 mV versus NHE for the [4Fe-4S]3+/2+ couple of Endo III, similar to Au. Without DNA modification, oxidative and reductive signals for the [4Fe-4S] cluster of Endo III are found on bare HOPG, allowing a direct comparison between DNA-bound and free redox potentials. These data indicate a shift of approximately -200 mV in the 3+/2+ couple upon binding of Endo III to DNA. This potential shift reflects a difference in affinity for DNA of more than 3 orders of magnitude between the oxidized 3+ and reduced 2+ protein and provides quantitative support for our model utilizing DNA-mediated charge transport to redistribute base excision repair enzymes in the vicinity of damaged DNA.

DNA↗
PubMed2006

Electrochemistry using self-assembled DNA monolayers on highly oriented pyrolytic graphite.

Duplex DNA functionalized with pyrene has been utilized to fabricate DNA-modified electrodes on highly oriented pyrolytic graphite (HOPG). Films have been characterized using AFM and radioactive labeling as well as electrochemically. The data obtained are consistent with a close-packed structure in the film with helices oriented in a nearly upright orientation, as seen earlier with the fabrication of thiol-tethered duplexes on gold. Also as on gold, we observe the reduction of DNA-bound intercalators in a DNA-mediated reaction. The reduction of the intercalator is attenuated in the presence of the single-base mismatches, CA and GT, independent of the sequence composition of the oligonucleotide. This sensitivity to single-base mismatches is enhanced when methylene blue reduction is coupled in an electrocatalytic cycle with ferricyanide. The extended potential range afforded by the HOPG surface has allowed us also to investigate the electrochemistry of previously inaccessible metallointercalators, Ru(bpy)2dppz2+ and Os(phen)2dppz2+, at the DNA-modified HOPG surface. These results support the application of DNA-modified HOPG as a convenient and reproducible surface for electrochemical DNA sensors using DNA-mediated charge transport.

Cross-Linking Reagents↗
PubMed2006

Site-specific DNA photocleavage by rhodium intercalators analyzed by MALDI-TOF mass spectrometry.

The DNA photocleavage reaction of mismatch-selective Rh complexes has been analyzed by MALDI-TOF mass spectrometry as well as gel electrophoresis analysis of radioactively tagged oligonucleotides. Analogous results are obtained with these two techniques showing site-specific cleavage neighboring the mismatch to yield primarily 5'- and 3'-phosphate termini. Additional intermediates and products are observed, however, using MALDI-TOF analysis. MALDI-TOF mass spectrometry is seen to be particularly powerful in the analysis of DNA cleavage by site-specific molecules. The method requires no radioactive labeling, only little material, and analysis can be accomplished within minutes. Moreover, this mass spectral analysis of DNA cleavage yields direct information regarding products rather than simply the base pair site of cleavage.

DNA↗
PubMed2006

A mismatch-selective bifunctional rhodium-Oregon Green conjugate: a fluorescent probe for mismatched DNA.

A fluorescent metallointercalator conjugate that selectively targets DNA base mismatches has been synthesized by coupling an organic fluorophore to a bulky Rh intercalator containing the chrysenequinone diimine ligand. Ion pairing between the cationic Rh and anionic fluorophore moieties dramatically quenches the fluorescence of the conjugate in solution and in the presence of matched DNA. However, in the presence of mismatched DNA, the fluorescence of the conjugate is increased >300%. This increase in fluorescence is attributed to the loss in intramolecular quenching associated with DNA binding; intercalation of the Rh moiety into the mismatched site can lead to electrostatic repulsion of the anionic fluorophore away from the DNA phosphate backbone and Rh. Denaturing PAGE experiments with 32P-labeled oligonucleotides indicate that the conjugate selectively binds the mismatched DNA with a binding affinity of 6 x 105 M-1 and, upon irradiation, cleaves the DNA backbone neighboring the mismatched site.

Autoradiography↗
PubMed2006

Electron trap for DNA-bound repair enzymes: a strategy for DNA-mediated signaling.

Despite a low copy number within the cell, base excision repair (BER) enzymes readily detect DNA base lesions and mismatches. These enzymes also contain [Fe4S4] clusters, yet a redox role for these iron cofactors had been unclear. Here, we provide evidence that BER proteins may use DNA-mediated redox chemistry as part of a signaling mechanism to detect base lesions. By using chemically modified bases, we show electron trapping on DNA in solution with bound BER enzymes by electron paramagnetic resonance (EPR) spectroscopy. We demonstrate electron transfer from two BER proteins, Endonuclease III (EndoIII) and MutY, to modified bases in DNA containing oxidized nitroxyl radical EPR probes. Electron trapping requires that the modified base is coupled to the DNA pi-stack, and trapping efficiency is increased when a noncleavable MutY substrate analogue is located distally to the trap. These results are consistent with DNA binding leading to the activation of the repair proteins toward oxidation. Significantly, these results support a mechanism for DNA repair that involves DNA-mediated charge transport.

Base Sequence↗
PubMed2005

Sequence dependence of charge transport through DNA domains.

Here we examine the photooxidation of two kinetically fast electron hole traps, N4-cyclopropylcytosine (CPC) and N2-cyclopropylamine-guanosine (CPG), incorporated in DNA duplexes of various sequence using different photooxidants. DNA oxidation studies are carried out either with noncovalently bound [Ru(phen)(dppz)(bpy')]3+ (dppz = dipyridophenazine) and [Rh(phi)2(bpy)]3+ (phi = phenanthrenequinone diimine) or with anthraquinone tethered to DNA. Because the cyclopropylamine-substituted bases decompose rapidly upon oxidation, their efficiency of decomposition provides a measure of relative hole localization. Consistent with a higher oxidation potential for CPC versus CPG in DNA, CPC decomposes with photooxidation by [Rh(phi)2(bpy)]3+, while CPG undergoes ring-opening both with photoexcited [Rh(phi)2(bpy)]3+ and with [Ru(phen)(dppz)(bpy')]3+. Anthraquinone-modified DNA assemblies of identical base composition but different base sequence are also probed. Single and double base substitutions within adenine tracts modulate CPC decomposition. In fact, the entire sequence within the DNA assembly is seen to govern CPC oxidation, not simply the bases intervening between CPC and the tethered photooxidant. These data are reconciled in the context of a mechanistic model of conformationally gated charge transport through delocalized DNA domains. Photooxidations of anthraquinone-modified DNA assemblies containing both CPC and CPG, but with varied distances separating the modified bases, point to a domain size of at least three bases. Our model for DNA charge transport is distinguished from polaron models. In our model, delocalized domains within the base pair stack form transiently based upon sequence-dependent DNA structure and dynamics. Given these results, DNA charge transport is indeed remarkably sensitive to DNA sequence and structure.

Base Sequence↗
PubMed2005

Reductive and oxidative DNA damage by photoactive platinum(II) intercalators.

Several photoactive platinum alpha-diimine intercalators have been prepared to develop new probes of DNA oxidation and reduction chemistry. Five water-soluble bis(mes')Pt(II) complexes (mes' = N,N,N,3,5-pentamethylaniline) with various aromatic alpha-diimine ligands (dppz = dipyridophenazine, np = naphtha[2,3-f][1,omega]phenanthroline, CN-np = naphtho[2,3-f][1,10]phenanthroline-9-carbonitrile, CN(2)-np = naphtho[2,3-f][1,10]phenanthroline-9,14-dicarbonitrile, and bp = benzo-[f][1,10]phenanthroline) were synthesized. The complex [(np)Pt(mes')(2)]Cl(2) was also characterized by X-ray crystallography, and the crystal structure shows that the ortho-methyl groups of the mes' ligands conveniently block substitution at the vacant sites of platinum without overlapping with the intercalating alpha-diimine ligand. The Pt(II) complexes were found to have excited-state oxidation and reduction potentials of -0.6 to -1.0 and 1.0 to 1.5 V versus NHE, respectively, making them potent photoreductants as well as photooxidants. Many of the complexes are found to promote the photooxidation of N(2)-cyclopropyldeoxyguanosine (d(Cp)G). Photoexcited [(dppz)Pt(mes')(2)](2+) is found to be most efficient in this photooxidation, as well as in the photoreduction of N(4)-cyclopropylcytidine ((Cp)C); these modified nucleosides rapidly decompose in a ring-opening reaction upon oxidation or reduction. Photoexcited [(dppz)Pt(mes')(2)]Cl(2), upon intercalation into the DNA pi stack, is found, in addition, to promote reductive and oxidative damage within the DNA duplex, as is also probed using the kinetically fast electron and hole traps, (Cp)C and (Cp)G. These Pt complexes may therefore offer useful reactive tools to compare and contrast directly reductive and oxidative chemistry in double helical DNA.

Cations, Divalent↗
PubMed2005

DNA charge transport leading to disulfide bond formation.

Here, we show that DNA-mediated charge transport (CT) can lead to the oxidation of thiols to form disulfide bonds in DNA. DNA assemblies were prepared possessing anthraquinone (AQ) as a photooxidant spatially separated on the duplex from two SH groups incorporated into the DNA backbone. Upon AQ irradiation, HPLC analysis reveals DNA ligated through a disulfide. The reaction efficiency is seen to vary in assemblies containing intervening DNA mismatches, confirming that the reaction is DNA-mediated. Interestingly, one intervening mismatch near the thiols promotes an increase in efficiency, which we attribute to increased base dynamics. Hence, here, where the reaction is on the backbone rather than within the base stack, stacking perturbations do not necessarily lead to an inhibitory effect on DNA CT.

Base Sequence↗
PubMed2005

DNA electrochemistry through the base pairs not the sugar-phosphate backbone.

Using intercalated, covalently bound daunomycin as a redox probe, ground state charge transport in DNA films with a perturbation in base pair stacking was examined in comparison with breaks in the sugar-phosphate backbone. While the introduction of one or even two nicks in the sugar-phosphate backbone yields no detectable effect on electron transfer, a CA mismatch significantly attenuates the electron transfer yield. These results confirm that the base pair stack is the pathway for DNA-mediated charge transfer, not the sugar-phosphate backbone.

Base Pair Mismatch↗
PubMed2005

Electrically monitoring DNA repair by photolyase.

Cyclobutane pyrimidine dimers are the major DNA photoproducts produced upon exposure to UV radiation. If left unrepaired, these lesions can lead to replication errors, mutation, and cell death. Photolyase is a light-activated flavoenzyme that binds to pyrimidine dimers in DNA and repairs them in a reaction triggered by electron transfer from the photoexcited flavin cofactor to the dimer. Using gold electrodes modified with DNA duplexes containing a cyclobutane thymine dimer (T<>T), here we probe the electrochemistry of the flavin cofactor in Escherichia coli photolyase. Cyclic and square-wave voltammograms of photolyase deposited on these electrodes show a redox signal at 40 mV versus normal hydrogen electrode, consistent with electron transfer to and from the flavin in the DNA-bound protein. This signal is dramatically attenuated on surfaces where the pi-stacking of the DNA bases is perturbed by the presence of an abasic site below the T<>T, an indication that the redox pathway is DNA-mediated. DNA repair can, moreover, be monitored electrically. Exposure of photolyase on T<>T-damaged DNA films to near-UV/blue light leads to changes in the flavin signal consistent with repair, as confirmed by parallel HPLC experiments. These results demonstrate the exquisite sensitivity of DNA electrochemistry to perturbations in base pair stacking and the applicability of this chemistry to probe reactions of proteins with DNA.

Base Sequence↗
PubMed2005

DNA-bound redox activity of DNA repair glycosylases containing [4Fe-4S] clusters.

MutY and endonuclease III, two DNA glycosylases from Escherichia coli, and AfUDG, a uracil DNA glycosylase from Archeoglobus fulgidus, are all base excision repair enzymes that contain the [4Fe-4S](2+) cofactor. Here we demonstrate that, when bound to DNA, these repair enzymes become redox-active; binding to DNA shifts the redox potential of the [4Fe-4S](3+/2+) couple to the range characteristic of high-potential iron proteins and activates the proteins toward oxidation. Electrochemistry on DNA-modified electrodes reveals potentials for Endo III and AfUDG of 58 and 95 mV versus NHE, respectively, comparable to 90 mV for MutY bound to DNA. In the absence of DNA modification of the electrode, no redox activity can be detected, and on electrodes modified with DNA containing an abasic site, the redox signals are dramatically attenuated; these observations show that the DNA base pair stack mediates electron transfer to the protein, and the potentials determined are for the DNA-bound protein. In EPR experiments at 10 K, redox activation upon DNA binding is also evident to yield the oxidized [4Fe-4S](3+) cluster and the partially degraded [3Fe-4S](1+) cluster. EPR signals at g = 2.02 and 1.99 for MutY and g = 2.03 and 2.01 for Endo III are seen upon oxidation of these proteins by Co(phen)(3)(3+) in the presence of DNA and are characteristic of [3Fe-4S](1+) clusters, while oxidation of AfUDG bound to DNA yields EPR signals at g = 2.13, 2.04, and 2.02, indicative of both [4Fe-4S](3+) and [3Fe-4S](1+) clusters. On the basis of this DNA-dependent redox activity, we propose a model for the rapid detection of DNA lesions using DNA-mediated electron transfer among these repair enzymes; redox activation upon DNA binding and charge transfer through well-matched DNA to an alternate bound repair protein can lead to the rapid redistribution of proteins onto genome sites in the vicinity of DNA lesions. This redox activation furthermore establishes a functional role for the ubiquitous [4Fe-4S] clusters in DNA repair enzymes that involves redox chemistry and provides a means to consider DNA-mediated signaling within the cell.

Archaeal Proteins↗
PubMed2005

Protein-DNA charge transport: redox activation of a DNA repair protein by guanine radical.

DNA charge transport (CT) chemistry provides a route to carry out oxidative DNA damage from a distance in a reaction that is sensitive to DNA mismatches and lesions. Here, DNA-mediated CT also leads to oxidation of a DNA-bound base excision repair enzyme, MutY. DNA-bound Ru(III), generated through a flash/quench technique, is found to promote oxidation of the [4Fe-4S](2+) cluster of MutY to [4Fe-4S](3+) and its decomposition product [3Fe-4S](1+). Flash/quench experiments monitored by EPR spectroscopy reveal spectra with g = 2.08, 2.06, and 2.02, characteristic of the oxidized clusters. Transient absorption spectra of poly(dGC) and [Ru(phen)(2)dppz](3+) (dppz = dipyridophenazine), generated in situ, show an absorption characteristic of the guanine radical that is depleted in the presence of MutY with formation instead of a long-lived species with an absorption at 405 nm; we attribute this absorption also to formation of the oxidized [4Fe-4S](3+) and [3Fe-4S](1+) clusters. In ruthenium-tethered DNA assemblies, oxidative damage to the 5'-G of a 5'-GG-3' doublet is generated from a distance but this irreversible damage is inhibited by MutY and instead EPR experiments reveal cluster oxidation. With ruthenium-tethered assemblies containing duplex versus single-stranded regions, MutY oxidation is found to be mediated by the DNA duplex, with guanine radical as an intermediate oxidant; guanine radical formation facilitates MutY oxidation. A model is proposed for the redox activation of DNA repair proteins through DNA CT, with guanine radicals, the first product under oxidative stress, in oxidizing the DNA-bound repair proteins, providing the signal to stimulate DNA repair.

DNA↗
PubMed2004

Long-range oxidative damage to cytosines in duplex DNA.

Charge transport (CT) through DNA has been found to occur over long molecular distances in a reaction that is sensitive to intervening structure. The process has been described mechanistically as involving diffusive charge-hopping among low-energy guanine sites. Using a kinetically fast electron hole trap, N(4)-cyclopropylcytosine ((CP)C), here we show that hole migration must involve also the higher-energy pyrimidine bases. In DNA assemblies containing either [Rh(phi)(2)(bpy')](3+) or an anthraquinone derivative, two high-energy photooxidants, appreciable oxidative damage at a distant (CP)C is observed. The damage yield is modulated by lower-energy guanine sites on the same or complementary strand. Significantly, the efficiency in trapping at (CP)C is equivalent to that at N(2)-cyclopropylguanosine ((CP)G). Indeed, even when (CP)G and (CP)C are incorporated as neighboring bases on the same strand, their efficiency of photodecomposition is comparable. Thus, CT is not simply a function of the relative energies of the isolated bases but instead may require orbital mixing among the bases. We propose that charge migration through DNA involves occupation of all of the DNA bases with radical delocalization within transient structure-dependent domains. These delocalized domains may form and break up transiently, facilitating and limiting CT. This dynamic delocalized model for DNA CT accounts for the sensitivity of the process to sequence-dependent DNA structure and provides a basis to reconcile and exploit DNA CT chemistry and physics.

Anthraquinones↗