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

Biological contexts for DNA charge transport chemistry

Many experiments have now shown that double helical DNA can serve as a conduit for efficient charge transport (CT) reactions over long distances in vitro. These results prompt the consideration of biological roles for DNA-mediated CT. DNA CT has been demonstrated to occur in biologically relevant environments such as within the mitochondria and nuclei of HeLa cells as well as in isolated nucleosomes. In mitochondria, DNA damage that results from CT is funneled to a crucial regulatory element. Thus, DNA CT provides a strategy to funnel damage to particular sites in the genome. DNA CT might also be important in long-range signaling to DNA-bound proteins. Both DNA repair proteins, containing Fe-S clusters, and the transcription factor, p53, which is regulated through thiol-disulfide switches, can be oxidized from a distance through DNA-mediated CT. These observations highlight a means through which oxidative stress may be chemically signaled in the genome over long distances through CT from guanine radicals to DNA-bound proteins. Moreover, DNA-mediated CT may also play a role in signaling among DNA-binding proteins, as has been proposed as a mechanism for how DNA repair glycosylases more efficiently detect lesions inside the cell

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

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 [3Fe4S](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

Facile and E-Selective Intramolecular Ring-Closing Metathesis Reactions in 3_(10)-Helical Peptides: A 3D Structural Study

The ring-closing metathesis reaction can be used to cross-link allylated serine residues situated at the i and i + 3 positions in 3_(10)-helical peptides containing the helicogenic amino acid, α-aminoisobutyric acid (Aib). An octapeptide with the sequence Boc-Aib-Aib-Aib-Ser(Al)-Aib-Aib-Ser(Al)-Aib-OMe was found to undergo a facile and >20:1 E-selective ring-closing metathesis (RCM) reaction catalyzed by the Grubbs second-generation catalyst to yield an 18-membered macrocycle. The formation of this cross-link does not significantly disturb the peptide's native 3_(10)-helicity, as judged by an X-ray diffraction study of the acyclic diene, the E-olefin RCM product, and its hydrogenated derivative. A heptapeptide system with the sequence Boc-Val-Ser(Al)-Leu-Aib-Ser(Al)-Val-Leu-OMe also underwent an efficient RCM reaction, albeit with diminished E-selectivity. It is apparent from these studies that a minimal, RCM-derived, macrocyclic constraint can be readily incorporated into 3_(10)-helical peptides

Hydrogen Donation but not Abstraction by a Tyrosine (Y68) During Endoperoxide Installation by Verruculogen Synthase (FtmOx1)

Hydrogen-atom transfer (HAT) from a substrate carbon to an iron(IV)-oxo (ferryl) intermediate initiates a diverse array of enzymatic transformations. For outcomes other than hydroxylation, coupling of the resultant carbon radical and hydroxo ligand (oxygen rebound) must generally be averted. A recent study of FtmOx1, a fungal iron(II)- and 2-(oxo)glutarate-dependent oxygenase that installs the endoperoxide of verruculogen by adding O_2 between carbons 21 and 27 of fumitremorgin B, posited that tyrosine (Tyr or Y) 224 serves as HAT intermediary to separate the C21 radical (C21•) and Fe(III)–OH HAT products and prevent rebound. Our reinvestigation of the FtmOx1 mechanism revealed, instead, direct HAT from C21 to the ferryl complex and surprisingly competitive rebound. The C21-hydroxylated (rebound) product, which undergoes deprenylation, predominates when low [O_2] slows C21•–O_2 coupling in the next step of the endoperoxidation pathway. This pathway culminates with addition of the C21–O–O• peroxyl adduct to olefinic C27 followed by HAT to the C26• from a Tyr. The last step results in sequential accumulation of Tyr radicals, which are suppressed without detriment to turnover by inclusion of the reductant, ascorbate. Replacement of each of four candidates for the proximal C26 H• donor (including Y224) with phenylalanine (F) revealed that only the Y68F variant (i) fails to accumulate the first Tyr• and (ii) makes an altered major product, identifying Y68 as the donor. The implied proximities of C21 to the iron cofactor and C26 to Y68 support a new docking model of the enzyme–substrate complex that is consistent with all available data

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