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

Abstract

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.

Scheme 1.

Schematic illustration of the flash-quench technique used to generate Ru(III) in situ and subsequently to oxidize DNA-bound MutY. Back electron transfer reactions are in gray.

Fig. 1.

EPR spectroscopy at 10 K of DNA samples after irradiation of [Ru(phen)₂dppz]²⁺ (25 μM) with [Co(NH₃)₅Cl]²⁺ (125 μM) as quencher and poly(dGC) (1 mM bp) with and without MutY (50 μM) (A); poly(dAT) (1 mM bp) with and without MutY (50 μM) (B); and poly(dGC) (1 mM bp) with native MutY or C199H mutant (50 μM) (C).

Fig. 2.

Time-resolved transient absorption data for Ru(phen)₂(dppz)²⁺ (20 μM) bound to poly(dGC) (1 mM bp) quenched by [Ru(NH₃)₆]³⁺ (0.4 mM) with MutY (20 μM). Shown is the absorption difference spectrum of the long-lived transient with data averaged over four experiments. (Inset) Transient absorption at 405 nm in the presence (red) and absence (green) of MutY bound to poly(dGC) or without DNA (black).

Fig. 3.

Autoradiogram after denaturing PAGE of ³²P-5′-TTGGAATTATAATTTATAATATTAAATATT-3′ after oxidation of the ruthenium-tethered oligonucleotide duplex by flash/quench. Lanes shown are Maxam-Gilbert sequencing reactions for C + T and A + G. respectively. Lanes 1–5: Ru-DNA irradiated in the presence of cobalt quencher and 8, 6, 4, 2, or 0 μM MutY. Lane 6: Ru-DNA irradiated with 4 μM MutY but no quencher. Lane 7: Ru-DNA without MutY or quencher. Lane 8: DNA irradiated without Ru-tethered strand. Concentrations were [DNA] = 4 μM and [Co(NH₃)₅Cl]²⁺ = 200 μM. Irradiations were for 15 min. Reactions were carried out in 5 mM sodium phosphate, 50 mM NaCl, pH 7.

Fig. 4.

EPR spectroscopy at 10 K of ruthenium-tethered DNA duplexes (25 μM, fully or partially hybridized) with MutY (50 μM) after irradiation in the presence of quencher {[Co(NH₃)₅Cl]²⁺, 125 μM}.

Fig. 5.

Model for detection strategy for BER enzymes using DNA-mediated CT stimulated by guanine radicals. The guanine radicals, formed under oxidative stress, are reduced and hence repaired through DNA-mediated electron transfer from the BER enzyme (above). Oxidation of the repair protein then drives CT to an alternate repair protein bound at a distal site, thereby promoting the redistribution of DNA repair proteins on genomic sites. Because no DNA CT can proceed through intervening lesions, the proteins are preferentially redistributed onto sites near lesions (below). Thus guanine radicals, in oxidizing the DNA-bound repair proteins, and driving the redistribution, provide a signal to stimulate DNA repair.