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Protein–DNA charge transport: Redox activation of a DNA repair protein by guanine radical
- Eylon Yavin†,
- Amie K. Boal†,
- Eric D. A. Stemp†,
- Elizabeth M. Boon†,
- Alison L. Livingston‡,
- Valerie L. O'Shea‡,
- Sheila S. David‡,§, and
- Jacqueline K. Barton†,§
- †Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125; and ‡Department of Chemistry, University of Utah, Salt Lake City, UT 84112
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Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA (received for review December 16, 2004)
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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.
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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).
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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).
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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.
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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}.
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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.
Footnotes
- Copyright © 2005, The National Academy of Sciences
