Oxidation by DNA charge transport damages conserved sequence block II, a regulatory element in mitochondrial DNA

Sites of oxidative damage in mitochondrial DNA have been identified on the basis of DNA-mediated charge transport. Our goal is to understand which sites in mitochondrial DNA are prone to oxidation at long range and whether such oxidative damage correlates with cancerous transformation. Here we show that a primer extension reaction can be used to monitor directly oxidative damage to authentic mitochondrial DNA through photoreactions with a rhodium intercalator. The complex [Rh(phi)2bpy]Cl3 (phi = 9,10-phenanthrenequinone diimine) binds to DNA without sequence specificity and, upon photoactivation, either promotes strand breaks directly at the binding site or promotes one-electron oxidative damage; comparing the sites of base oxidation to direct strand breaks reveals the oxidative damage that arises from a distance through DNA-mediated charge transport. Significantly, base oxidation by charge transport overlaps with known mutational hot spots associated with cancers at nucleotides surrounding positions 263 and 303; the latter is known as conserved sequence block II and is vital to DNA replication. Since DNA base oxidation at conserved sequence block II should weaken the ability of damaged mitochondrial genomes to be replicated, DNA-mediated charge transport may provide a protection mechanism for excluding damaged DNA.     

DNA oxidation by charge transport in mitochondria

Sites of oxidative DNA damage in functioning mitochondria have been identified using a rhodium photooxidant as a probe. Here we show that a primer extension reaction can be used to monitor oxidative DNA damage directly in functioning mitochondria after photoreaction with a rhodium intercalator that penetrates the intact mitochondrial membrane. The complex [Rh(phi)2bpy]Cl3 (phi = 9,10-phenanthrenequinonediimine) binds to DNA within the mitochondria and, upon irradiation, initiates DNA oxidation reactions. Significantly, piperidine treatment of the mitochondria leads to protein-dependent primer extension stops spaced every approximately 20 base pairs. Hence, within the mitochondria, the DNA is well covered and packaged by proteins. Photolysis of the mitochondria containing [Rh(phi)2bpy]3+ leads to oxidative DNA damage at positions 260 and 298; both are mutational hot spots associated with cancers. The latter position is the 5'-nucleotide of conserved sequence block II and is critical to replication of the mitochondrial DNA. The oxidative damage is found to be DNA-mediated, utilizing a charge transport mechanism, as the Rh binding sites are spatially separated from the oxidation-prone regions. This long-range DNA-mediated oxidation occurs despite protein association. Indeed, the oxidation of the mitochondrial DNA leads not only to specific oxidative lesions, but also to a corresponding change in the protein-induced stops in the primer extension. Mitochondrial DNA damage promotes specific changes in protein-DNA contacts and is thus sensed by the mitochondrial protein machinery.     

DNA-mediated charge transport as a probe of MutY/DNA interaction

MutY is an Escherichia coli DNA repair enzyme that binds to 8-oxo-G:A and G:A mismatches and catalyzes the deglycosylation of the mismatched 2'-deoxyadenosine. We have applied DNA-mediated charge transport to probe the interaction of MutY with its DNA substrate. Oligonucleotides synthesized with a tethered rhodium intercalator and guanine doublets placed before and after the MutY binding site are used to assay for base flipping activity by MutY. On the basis of this assay, we find no evidence that MutY uses progressive base flipping as a means to find its binding site; protein binding does not perturb long-range DNA charge transport. DNA-mediated charge transport can be utilized to promote protein-DNA cross-linking from a distance. Long-range oxidation of 8-oxo-G within the MutY binding site using tethered rhodium intercalators promoted cross-linking and yielded information on MutY side chains that interact with this base. On the basis of photooxidative cross-linking of the wild type but not K142A mutant, it is evident that, within the protein complex, lysine 142 makes important contacts with 8-oxo-G.     

Electrochemical detection of lesions in DNA

Electrochemical DNA-based sensors that exploit the inherent sensitivity of DNA-mediated charge transport (CT) to base pair stacking perturbations are capable of detecting base pair mismatches and some common base damage products. Here, using DNA-modified gold electrodes, monitoring the electrocatalytic reduction of DNA-bound methylene blue, we examine a wide range of base analogues and DNA damage products. Among those detected are base damage products O4-methyl-thymine, O6-methyl-guanine, 8-oxo-guanine, and 5-hydroxy-cytosine, as well as a therapeutic base, nebularine. The efficiency of DNA-mediated CT is found not to depend on the thermodynamic stability of the helix. However, general trends in how base modifications affect CT efficiency are apparent. Modifications to the hydrogen bonding interface in Watson-Crick base pairs yields a substantial loss in CT efficiency, as does added steric bulk. Base structure modifications that may induce base conformational changes also appear to attenuate CT in DNA as do those that bury hydrophilic groups within the DNA helix. Addition and subtraction of methyl groups that do not disrupt hydrogen bonding interactions do not have a large effect on CT efficiency. This sensitive detection methodology based upon DNA-mediated CT may have utility in diagnostic applications and implicates DNA-mediated CT as a possible damage detection mechanism for DNA repair enzymes.     

Long-range guanine oxidation in DNA restriction fragments by a triplex-directed naphthalene diimide intercalator

Naphthalene diimide (NDI), a powerful oxidant that binds avidly to DNA by intercalation, is seen to damage the 5' guanine of 5'-GG-3' sites by photoactivated charge transport through DNA. When covalently tethered to the center of a triplex-forming oligonucleotide and delivered by triplex formation within a pyrimidine.purine-pyrimidine motif to a specific site on a restriction fragment, NDI can photooxidize guanine over at least 25-38 bp in each direction from the site of binding. Charge migration occurs in both directions from the NDI intercalator and on both DNA strands of the target, but the oxidation is significantly more efficient to the 3' side of the triplex. NDI and octahedral rhodium intercalators, when tethered directly to the 5' terminus of the triplex-forming strand as opposed to the center, generate significant amounts of oxidative damage only in the immediate vicinity of the intercalation site. Given that long-range charge transport depends on DNA stacking, these results suggest that the base stack is distorted at the 5' end of the triplex region in the duplex-triplex junction. Targeting of photooxidative damage by triplex formation extends our previous studies of long-range charge transport to significantly longer DNA sequences through a strategy that does not require covalent attachment of the photooxidant to the DNA being probed. Moreover, triplex targeting of oxidative damage provides for the first time a typical distance distribution for genomic charge transport of approximately 200 A around the oxidant.     

Charge transport through DNA four-way junctions

Long range oxidative damage as a result of charge transport is shown to occur through single crossover junctions assembled from four semi-complementary strands of DNA. When a rhodium complex is tethered to one of the arms of the four-way junction assembly, thereby restricting its intercalation into the π-stack, photo-induced oxidative damage occurs to varying degrees at all guanine doublets in the assembly, though direct strand scission only occurs at the predicted site of intercalation. In studies where the Mg²⁺ concentration was varied, so as to perturb base stacking at the junction, charge transport was found to be enhanced but not to be strongly localized to the arms that preferentially stack on each other. These data suggest that the conformations of four-way junctions can be relatively mobile. Certainly, in four-way junctions charge transport is less discriminate than in the more rigidly stacked DNA double crossover assemblies.     

Charge transport in DNA

The base pair stack within double helical DNA provides an effective medium for charge transport. The DNA pi-stack mediates oxidative DNA damage over long molecular distances in a reaction that is exquisitely sensitive to the sequence-dependent conformation and dynamics of DNA. A mixture of tunneling and hopping mechanisms have been proposed to account for this long-range chemistry, which is gated by dynamical variations within the stack. Electrochemical sensors have also been developed, based upon the sensitivity of DNA charge transport to base pair stacking, and these sensors provide a completely new approach to diagnosing single base mismatches in DNA and monitoring protein-DNA interactions electrically. DNA charge transport, furthermore, may play a role within the cell and, indeed, oxidative damage to DNA from a distance has been demonstrated in the cell nucleus. As a result, the biological consequences of and opportunities for DNA-mediated charge transport now require consideration.     

DNA repair: models for damage and mismatch recognition

Maintaining the integrity of the genome is critical for the survival of any organism. To achieve this, many families of enzymatic repair systems which recognize and repair DNA damage have evolved. Perhaps most intriguing about the workings of these repair systems is the actual damage recognition process. What are the chemical characteristics which are common to sites of nucleic acid damage that DNA repair proteins may exploit in targeting sites? Importantly, thermodynamic and kinetic principles, as much as structural factors, make damage sites distinct from the native DNA bases, and indeed, in many cases, these are the features which are believed to be exploited by repair enzymes. Current proposals for damage recognition may not fulfill all of the demands required of enzymatic repair systems given the sheer size of many genomes, and the efficiency with which the genome is screened for damage. Here we discuss current models for how DNA damage recognition may occur and the chemical characteristics, shared by damaged DNA sites, of which repair proteins may take advantage. These include recognition based upon the thermodynamic and kinetic instabilities associated with aberrant sites. Additionally, we describe how small changes in base pair structure can alter also the unique electronic properties of the DNA base pair pi-stack. Further, we describe photophysical, electrochemical, and biochemical experiments in which mismatches and other local perturbations in structure are detected using DNA-mediated charge transport. Finally, we speculate as to how this DNA electron transfer chemistry might be exploited by repair enzymes in order to scan the genome for sites of damage.     

DNA-mediated electrochemistry

The base pair stack of DNA has been demonstrated as a medium for long-range charge transport chemistry both in solution and at DNA-modified surfaces. This chemistry is exquisitely sensitive to structural perturbations in the base pair stack as occur with lesions, single base mismatches, and protein binding. We have exploited this sensitivity for the development of reliable electrochemical assays based on DNA charge transport at self-assembled DNA monolayers. Here, we discuss the characteristic features, applications, and advantages of DNA-mediated electrochemistry.     

DNA repair glycosylases with a [4Fe-4S] cluster: a redox cofactor for DNA-mediated charge transport?

The [4Fe-4S] cluster is ubiquitous to a class of base excision repair enzymes in organisms ranging from bacteria to man and was first considered as a structural element, owing to its redox stability under physiological conditions. When studied bound to DNA, two of these repair proteins (MutY and Endonuclease III from Escherichia coli) display DNA-dependent reversible electron transfer with characteristics typical of high potential iron proteins. These results have inspired a reexamination of the role of the [4Fe-4S] cluster in this class of enzymes. Might the [4Fe-4S] cluster be used as a redox cofactor to search for damaged sites using DNA-mediated charge transport, a process well known to be highly sensitive to lesions and mismatched bases? Described here are experiments demonstrating the utility of DNA-mediated charge transport in characterizing these DNA-binding metalloproteins, as well as efforts to elucidate this new function for DNA as an electronic signaling medium among the proteins.     

Probing DNA charge transport with metallointercalators

A wide range of experiments have emerged recently regarding charge transport through DNA, including spectroscopic studies of rates of DNA-mediated electron transfer and biochemical studies of DNA base oxidation over long distances. These experiments have, in turn, led to new proposals about the way in which charge moves through DNA and have prompted the consideration of physiological roles for DNA electron transfer. Importantly, metallointercalators have been key players in many of these experiments. Metallointercalators provide critical probes to examine the migration of charge through the DNA base stack.     

Coupling into the base pair stack is necessary for DNA-mediated electrochemistry

The electrochemistry of DNA films modified with different redox probes linked to DNA through saturated and conjugated tethers was investigated. Experiments feature two redox probes bound to DNA on two surfaces: anthraquinone (AQ)-modified uridines incorporated into thiolated DNA on gold (Au) and 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO)-modified uridines in pyrene-labeled DNA on highly oriented pyrolytic graphite (HOPG). The electrochemistry of these labels when incorporated into DNA has been examined in DNA films containing both well matched and mismatched DNA. DNA-mediated electrochemistry is found to be effective for the TEMPO probe linked with an acetylene linker but not for a saturated TEMPO connected through an ethylenediamine linker. For the AQ probe, DNA-mediated electrochemistry is found with an acetylene linker to uridine but not with an alkyl chain to the 5' terminus of the oligonucleotide. Large electrochemical signals and effective discrimination of intervening base mismatches are achieved for the probes connected through the acetylene linkages, while probes connected through saturated linkages exhibit small electrochemical signals associated only with direct surface to probe charge transfer and poor mismatch discrimination. Thus DNA electrochemistry with these probes is dramatically influenced by the chemical nature of their linkage to DNA. These results highlight the importance of effective coupling into the pi-stack for long-range DNA-mediated electrochemistry.     

Charge transport in DNA duplex/quadruplex conjugates

DNA conjugates containing adjacent duplex and guanine quadruplex assemblies have been designed to explore charge transport into quadruplex architectures. The quadruplex assemblies have been characterized structurally using circular dichroism and by assaying for chemical protection. Using an intercalating rhodium photooxidant, noncovalently bound or tethered to the duplex end, oxidizing radicals are found to be trapped in the folded quadruplex. Damage is observed almost exclusively at the external tetrads of the quadruplex. Little damage of the center tetrad is observed, due most likely to lowered efficiency of radical trapping within the quadruplex core. This pattern of damage is distinct from that observed for repetitive G sequences within duplex DNA. The data indicate, furthermore, that in the conjugates examined, the guanine quadruplex provides a more effective trap than a 5'-GG-3' guanine doublet within duplex DNA. Within these assemblies, sufficient base-base overlap must exist at the duplex/quadruplex junction to allow for charge migration. This funneling of damage to the quadruplex, as well as the unique pattern of damage within the quadruplex, requires consideration with respect to the analysis of oxidative DNA damage within the cell.     

Poly(deoxyadenylic-deoxythymidylic acid) damage by radiolytically activated neocarzinostatin

The anaerobic reaction of poly(deoxyadenylic-deoxythymidylic acid) with neocarzinostatin activated by the carboxyl radical CO2-, an electron donor generated from gamma-ray radiolysis of nitrous oxide saturated formate buffer, has been characterized. DNA damage includes base release and strand breaks. Few strand breaks are formed prior to alkaline treatment; they bear 3'-phosphoryl termini. In contrast, most (66%) of the base release occurs spontaneously. DNA damage is highly (95%) specific for thymidine sites. Neither DNA-drug covalent adduct nor nucleoside 5'-aldehyde, which are major products in the DNA-nicking reaction initiated by mercaptans and oxygen, is formed in this reaction. Data are presented to show that the CO2(-)-activated neocarzinostatin intermediate is a short-lived free radical able to abstract hydrogen atoms from the C-1' and C-5' positions of deoxyribose. Attack occurs mostly (68%) at the C-1' position, producing a lesion whose properties are consistent with those of (oxidized) apyrimidinic sites.     

Redmond Red as a redox probe for the DNA-mediated detection of abasic sites

Redmond Red, a fluoropore containing a redox-active phenoxazine core, has been explored as a new electrochemical probe for the detection of abasic sites in double-stranded DNA. The electrochemical behavior of Redmond Red-modified DNA at gold surfaces exhibits stable, quasi-reversible voltammetry with a midpoint potential centered around -50 mV versus NHE. Importantly, with Redmond Red positioned opposite an abasic site within the DNA duplex, the electrochemical response is significantly enhanced compared to Redmond Red positioned across from a base. Redmond Red, reporting only if well-stacked in the duplex, represents a sensitive probe to detect abasic sites electrochemically in a DNA-mediated reaction.     

Efficiency of repair of an abasic site within DNA clustered damage sites by mammalian cell nuclear extracts

Ionizing radiation induces clustered DNA damage sites which have been shown to challenge the repair mechanism(s) of the cell. Evidence demonstrating that base excision repair is compromised during the repair of an abasic (AP) site present within a clustered damage site is presented. Simple bistranded clustered damage sites, comprised of either an AP-site and 8-oxoG or two AP-sites, one or five bases 3' or 5' to each other, were synthesized in oligonucleotides, and repair was carried out in xrs5 nuclear extracts. The rate of repair of an AP-site when present opposite 8-oxoG is reduced by up to 2-fold relative to that when an AP-site is present as an isolated lesion. The mechanism of repair of the AP-site shows asymmetry, depending on its position relative to 8-oxoG on the opposite strand. The AP-site is rejoined by short-patch base excision repair when the lesions are 5' to each other, whereas when the lesions are 3' to one another, rejoining of the AP-site occurs by both long-patch and short-patch repair processes. The major stalling of repair occurs at the DNA ligase step. 8-OxoG and an AP-site present within a cluster are processed sequentially, limiting the formation of double-strand breaks to <4%. In contrast, when two AP-sites are contained within the clustered DNA damage site, both AP-sites are incised simultaneously, giving rise to double-strand breaks. This study provides new insight into understanding the processes that lead to the biological consequences of radiation-induced DNA damage and ultimately tumorigenesis.     

8-OxoG retards the activity of the ligase III/XRCC1 complex during the repair of a single-strand break, when present within a clustered DNA damage site

Ionising radiation produces clustered DNA damage. Recent studies have established that the efficiency of excision of a lesion within clustered damage sites is reduced. This study presents evidence that the repair of clustered DNA damage is compromised, relative to that of the isolated lesions, since the lifetime of both lesions is extended by up to eight fold. Simple clustered damage sites, comprised of a single-strand break, one or five bases 3' or 5' to 8-oxoG on the opposite strand, were synthesised in oligonucleotides and repair carried out in XRS5 nuclear extracts. The rate of repair of the single-strand break within these clustered damage sites is reduced, mainly due to inhibition of the DNA ligase III/XRCC1 complex. The single-strand break, present as an isolated lesion, is repaired by short-patch base excision repair, however the mechanism of repair of the single-strand break within the clustered damage site is asymmetric. When the lesions are 5' to each other, the single-strand break is rejoined by short-patch repair whereas the rejoining of the single-strand break occurs by long-patch type repair when the lesions are 3' to one another. The retardation of DNA ligase III/XRCC1 complex, following addition of one base, is responsible for the initiation of long-patch base excision repair when the lesions are 3' to each other. The lesions within the cluster are processed sequentially, the single-strand break being repaired before excision of 8-oxoG, limiting the formation of double-strand breaks to <2%. Stalled processing of clustered DNA damage is suggested to have implications for mutation induction by radiation.