Locating the uracil-5-yl radical formed upon photoirradiation of 5-bromouracil-substituted DNA

In a previous study, we found that 2-deoxyribonolactone is effectively generated in the specific 5-bromouracil ((Br)U)-substituted sequence 5′-(G/C)[A](n) (= 1,2)(Br)U(Br)U-3′ and proposed that a formed uracil-5-yl radical mainly abstracts the C1′ hydrogen from the 5′-side of (Br)U(Br)U under 302-nm irradiation condition. In the present work, we performed photoirradiation of (Br)U-substituted DNA in the presence of a hydrogen donor, tetrahydrofuran, to quench the uracil-5-yl radical to uracil and then subjected the sample to uracil DNA glycosylase digestion. Slab gel sequence analysis indicated that uracil residues were formed at the hot-spot sequence of 5′-(G/C)[A](n) (= 1,2)(Br)U(Br)U-3′ in 302-nm irradiation of (Br)U-substituted DNA. Furthermore, we found that the uracil residue was also formed at the reverse sequence 5′-(Br)U(Br)U[A](n) (= 1,2)(G/C)-3′, which suggests that both 5′-(G/C)[A](n) (= 1,2)(Br)U(Br)U-3′ and 5′-(Br)U(Br)U[A](n) (= 1,2)(G/C)-3′ are hot-spot sequences for the formation of the uracil-5-yl radical.