Repair of oxidative DNA damage by amino acids

Abstract

Guanyl radicals, the product of the removal of a single electron from guanine, are produced in DNA by the direct effect of ionizing radiation. We have produced guanyl radicals in DNA by using the single electron oxidizing agent (SCN)2-, itself derived from the indirect effect of ionizing radiation via thiocyanate scavenging of OH. We have examined the reactivity of guanyl radicals in plasmid DNA with the six most easily oxidized amino acids cysteine, cystine, histidine, methionine, tryptophan and tyrosine and also simple ester and amide derivatives of them. Cystine and histidine derivatives are unreactive. Cysteine, methionine, tyrosine and particularly tryptophan derivatives react to repair guanyl radicals in plasmid DNA with rate constants in the region of approximately 10(5), 10(5), 10(6) and 10(7) dm3 mol(-1) s(-1), respectively. The implication is that amino acid residues in DNA binding proteins such as histones might be able to repair by an electron transfer reaction the DNA damage produced by the direct effect of ionizing radiation or by other oxidative insults.

Figure 1

Loss of supercoiled plasmid with increasing dose of γ-radiation. Aliquots of a solution containing plasmid pHAZE (25 µg ml^–1), sodium phosphate (5 × 10^–3 mol dm^–3, pH 7.0), sodium thiocyanate (10^–3 mol dm^–3), sodium perchlorate (1.1 × 10^–1 mol dm^–3) and tyrosine methyl ester [3 × 10^–7 (open inverted triangle), 7.5 × 10^–7 (open triangle), 1.5 × 10^–6 (open diamond), 3 × 10^–6 (open circle) or 5 × 10^–6 (open and closed squares) mol dm^–3] were irradiated under aerobic conditions with ¹³⁷Ce γ-rays (662 keV) at dose rates between 1.7 × 10^–3 and 9.0 × 10^–2 Gy s^–1. After irradiation, the solutions were incubated for 30 min at 37°C with FPG at a concentration of 0 (closed square) or 5 µg ml^–1 (open symbols). The fraction of supercoiled plasmid remaining after each radiation dose and incubation was determined using agarose gel electrophoresis. These six data sets are plotted together. Each is fitted to least mean squares straight lines of the form y = ce^–mx. From the slopes m of these fitted straight lines, the doses D₀ and SSB yields for the six irradiation and incubation conditions are: open inverted triangle, 0.234 Gy, 1.59 × 10^–2 µmol J^–1; open triangle, 0.463 Gy, 8.04 × 10^–3 µmol J^–1; open diamond, 0.971 Gy, 3.84 × 10^–3 µmol J^–1; open circle, 1.94 Gy, 1.92 × 10^–3 µmol J^–1; open square, 3.41 Gy, 1.09 × 10^–3 µmol J^–1; closed square, 22.5 Gy, 1.66 × 10^–4 µmol J^–1.

Figure 2

Effect of tyrosine methyl ester concentration during irradiation on the SSB yield after incubation with or without FPG. Plasmid pHAZE was γ-irradiated in the presence of various concentrations of tyrosine methyl ester and the resulting SSB yield was determined (see Fig. 1) after incubation in the presence (open square) or absence (closed square) of FPG.

Figure 3

Reciprocal plot of the attenuation of the yield of FPG-sensitive sites, G(FPG), against the concentration of tyrosine methyl ester according to competition kinetics (see Discussion). The data set is fitted to a least mean squares straight line of the form y = mx + c. The slope m of the line is 1.8 × 10⁸ MJ dm³ mol^–2. See Discussion for an evaluation of the intercept c.