Thermally stable sites for electron capture in directly ionized DNA : free radicals produced by the net gain of hydrogen at C5/C6 of cytosine and thymine in crystalline oligodeoxynucleotides

Electron paramagnetic resonance (EPR) spectroscopy is used to study radical trapping in crystalline oligodeoxynucleotides exposed to 70 keV x-irradiation at 4 K and annealed to 240 K. The four oligomers studied were the Z form d(CGCACG:GCGTGC), two A forms, d(CCCTAGGG)2 and d(GTGCGCAC)2, and the B form d(CGCGAATTCGCG)2. In each of these oligomers, evidence was found for trapping of a cytosine radical formed by the net gain of a hydrogen at C6 and a proton at N3 (the Cyt(C6+H, N3+H+)+• radical). The data are consistent with the trapping of another cytosine radical formed by the net gain of hydrogen at C5 (the Cyt(C5+H, N3+H)+• or Cyt(C5+H)• radical). The well-known thymine radical formed by the net gain of hydrogen at C5 (Thy(C6+H)• radical) was observed in the Z- and B-form duplexes but not in the A-form duplexes. The relative yields of these three reduction species indicate that cytosine is comparable to, or better than, thymine as a stable trapping site for reductive damage. These three radicals, Cyt(C6+H, N3+H+)+•, Cyt(C5+H, N3+H)+•, and Thy(C6+H)•, account for 85% of the total irreversibly trapped electrons in samples irradiated at 4 K and annealed to 240 K. Extrapolation of these results to B-form DNA hydrated to 9 waters per nucleotide, x-irradiated at 4 K, and warmed to room temperature predicts end product yields of 0.04-0.06 µmol/J for 5,6-dihydrouracil and 0.03-0.05 µmol/J for 5,6-dihydrothymine.