DNA-mediated charge transport in redox sensing and signaling

Abstract

The transport of charge through the DNA base-pair stack offers a route to carry out redox chemistry at a distance. Here we describe characteristics of this chemistry that have been elucidated and how this chemistry may be utilized within the cell. The shallow distance dependence associated with these redox reactions permits DNA-mediated signaling over long molecular distances in the genome and facilitates the activation of redox-sensitive transcription factors globally in response to oxidative stress. The long-range funneling of oxidative damage to sites of low oxidation potential in the genome also may provide a means of protection within the cell. Furthermore, the sensitivity of DNA charge transport to perturbations in base-pair stacking, as may arise with base lesions and mismatches, may be used as a route to scan the genome for damage as a first step in DNA repair. Thus, the ability of double-helical DNA in mediating redox chemistry at a distance provides a natural mechanism for redox sensing and signaling in the genome.

Figure 1

Illustrative schematic of the generation and detoxification of hydrogen peroxide, an archetypical ROS, and some of its roles in mammalian redox signaling. In this diagram, we see production of hydrogen peroxide in the mitochondria (bottom left) accompanied by its efficient detoxification in both the matrix and the cytosol (center left) and the consequences of excess H₂O₂ reacting with ferrous iron (bottom left, right). H₂O₂ is a possible intermediate in the receptor-mediated pathways that rely on NOX activation. (top) Inside the nucleus, many transcription factors require further reduction, such as by APE-1 (right). The blue dashed lines represent the equilibration between the bulk concentration of H₂O₂ in the cell, and the local concentrations generated as part of signaling or pathological processes; that these processes are functionally distinct illustrates how difficult it is to deconvolute redox sensing and signaling pathways in mammals versus in yeast or prokaryotes. Poorly understood processes are represented as black dashes; in particular, the intermediate species in receptor-initiated ROS signaling are not well-characterized. Abbreviations: ecSOD, extracellular superoxide dismutase; NADPH, nicotinamide adenine dinucleotide phosphate; MAP3K, mitogen-activated protein kinase kinase kinase; MAP2K, mitogen-activated protein kinase kinase; NOX, NADPH oxidase; PTP, protein tyrosine phosphatase; Trx, thioredoxin; ASK-1, apoptosis signaling kinase; Tpx, thioredoxin peroxidase; Cat, catalase; JNK, c-jun N-terminal kinase; Gpx, glutathione peroxidase; Grd, glutathione reductase; MAO, monoamine oxidase; MnSOD, manganese superoxide dismutase; Acn, aconitase; APE-1, apurinic/apyrimidinic endonuclease. These redox pathways and their inter-relationships are discussed in detail in references 8-, , , and .

Figure 2

Three types of assemblies employed for studying DNA-mediated CT. An electrode is used to inject an electron and reduce a redox probe attached to the DNA (left). DNA-mediated CT is observed as the quenching mechanism of photoexcited [Ru(phen′)₂(dppz)]*^/2+ by [Rh(phi)₂(phen′)]³⁺ (top right). Both of these reactions occur at potentials insufficient to reduce or oxidize the DNA. A high energy photoexcited [Rh(phi)₂(phen′)]*^/3+ is employed that is competent to oxidize all the DNA bases (bottom right). Permanent chemical decomposition products are observed at 5′-GG-3′ steps. Hence, DNA can participate as both a mediator and as a reactant in CT.

Figure 3

DNA-mediated CT is sharply attenuated by the presence of a mismatch or other lesions that affect stacking. The accumulated current through the DNA is far less when oxidative lesions are present, including the physiologically relevant 8-oxoguanosine base-paired with adenosine and 5-hydroxycytidine base-paired with guanine.

Figure 4

DNA-mediated CT from a strongly oxidizing intercalated ruthenium complex to an intercalated lysine-tyrosine-lysine tripeptide (KYK) proceeds through a guanine radical intermediate. Oxidation of KYK leads to DNA-tyrosine cross-linking, while KWK is oxidized but does not form a covalent product with the DNA. This serves as a model system for the oxidation of DNA-bound proteins by radical species in DNA.

Figure 5

The conserved sequence block II (CSBII) serves as a checkpoint for mitochondrial DNA replication. Reactive oxygen species (ROS) introduce oxidative damage to the DNA, which we have shown to be funneled to the long polypurine region in CSBII, leading to oxidative decomposition of guanine to 8-oxoguanine at this site. This damage might interrupt specific CSBII-RNAse interactions that are required for replication.

Figure 6

SoxR is able to exploit DNA as an antenna for sensing oxidative stress conditions. Oxidative damage generates guanine radical intermediates, which migrate over long distances in DNA. These species oxidize SoxR, activating it to promote the transcription of soxS, inducing the oxidative stress response in E. coli.

Figure 7

Redox-active base excision repair (BER) enzymes can exploit DNA-mediated CT to rapidly assay regions of DNA for damage, such as oxidative lesions. The presence of a lesion serves as a barrier for DNA CT, leading to accumulation of BER proteins near the lesion. Importantly, BER enzymes of similar potential can assist each other in searching for damage. Here, the orange and blue proteins represent two different FeS cluster containing proteins of similar potential. In this model, guanine cation radical or another oxidative intermediate oxidizes a nearby BER FeS cluster to the tight-binding 3+ state (1). Binding of reduced protein nearby (2) allows self-exchange (3), following which the first protein, now in the 2+ state, has decreased affinity for DNA and can diffuse away (4). Steps 2-4 correspond to a net translation of 3+ protein on the DNA. This process is repeated when another reduced protein binds the DNA on the other side (5,7), followed by self-exchange and dissociation of the newly reduced protein (6,8). The DNA is left empty of the 3+, and hence strongly bound, protein (9). The directionality portrayed in (1-9) is illustrative, as the CT-mediated migration of 3+ protein is diffusive. When a lesion is present, steps 10-14 mirror steps 1-5. Reduced protein distal to the lesion, however, is unable to reduce the oxidized protein through DNA-mediated CT (15), increasing the residence of that particular 3+ protein near the lesion. CT-mediated migration of oxidized protein, as shown in the previous steps, can oxidize reduced protein on the distal side of the lesion (16), and 3+ protein accumulated near the lesion can slide along the DNA (17) and repair the lesion upon direct contact (18).

Figure 8

The delivery of radical holes induces dissociation of p53 from the promoter for GADD45, but not that for p21. DNA-mediated CT serves as a mechanism for efficient delivery of oxidation to p53, allowing a rapid redistribution from pro-repair to pro-apoptotic promoter sites.