Oxidation of Guanine in G, GG, and GGG Sequence Contexts by Aromatic Pyrenyl Radical Cations and Carbonate Radical Anions: Relationship between Kinetics and Distribution of Alkali-Labile Lesions

Oxidatively generated DNA damage induced by the aromatic radical cation of the pyrene derivative 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene (BPT), and by carbonate radicals anions, was monitored from the initial one-electron transfer, or hole injection step, to the formation of hot alkali-labile chemical end-products monitored by gel electrophoresis. The fractions of BPT molecules bound to double-stranded 20−35-mer oligonucleotides with noncontiguous guanines G and grouped as contiguous GG and GGG sequences were determined by a fluorescence quenching method. Utilizing intense nanosecond 355 nm Nd:YAG laser pulses, the DNA-bound BPT molecules were photoionized to BPT•+ radicals by a consecutive two-photon ionization mechanism. The BPT•+ radicals thus generated within the duplexes selectively oxidize guanine by intraduplex electron-transfer reactions, and the rate constants of these reactions follow the trend 5‘-..GGG.. > 5‘-..GG.. > 5‘-..G... In the case of CO3•- radicals, the oxidation of guanine occurs by intermolecular collision pathways, and the bimolecular rate constants are independent of base sequence context. However, the distributions of the end-products generated by CO3•- radicals, as well as by BPT•+, are base sequence context-dependent and are greater than those in isolated guanines at the 5‘-G in 5‘-...GG... sequences, and the first two 5‘- guanines in the 5‘-..GGG sequences. These results help to clarify the conditions that lead to a similar or different base sequence dependence of the initial hole injection step and the final distribution of oxidized, alkali-labile guanine products. In the case of the intermolecular one-electron oxidant CO3•-, the rate constant of hole injection is similar for contiguous and isolated guanines, but the subsequent equilibration of holes by hopping favors trapping and product formation at contiguous guanines, and the sequence dependence of these two phenomena are not correlated. In contrast, in the case of the DNA-bound oxidant BPT•+, the hole injection rate constants, as well as hole equilibration, exhibit a similar dependence on base sequence context, and are thus correlated to one another

Photosensitized Oxidative DNA Damage: From Hole Injection to Chemical Product Formation and Strand Cleavage

Oxidatively generated damage to DNA induced by a pyrenyl photosensitizer residue (Py) covalently attached to a guanine base in the DNA sequence context 5‘-d(CAT[G1Py]CG2TCCTAC) in aerated solutions was monitored from the initial one-electron transfer, or hole injection step, to the formation of chemical end-products monitored by HPLC, mass spectrometry, and high-resolution gel electrophoresis. Hole injection into the DNA was initiated by two-photon excitation of the Py residue with 355 nm laser pulses, thus producing the radical cation Py•+ and hydrated electrons; the latter are trapped by O2, thus forming the superoxide anion O2•-. The decay of the Py•+ radical is correlated with the appearance of the G•+/G(−H)• radical on microsecond time scales, and O2•- combines with guanine radicals at G1 to form alkali-labile 2,5-diamino-4H-imidazolone lesions (Iz1Py). Product formation in the modified strand is smaller by a factor of 2.4 in double-stranded than in single-stranded DNA. In double-stranded DNA, hot piperidine-mediated cleavage at G2 occurs only after G1Py, an efficient hole trap, is oxidized thus generating tandem lesions. An upper limit of hole hopping rates, khh 3 s-1 from G1•+−Py to G2 can be estimated from the known rates of the combination reaction of the G(−H)• and O2•- radicals. The formation of Iz products in the unmodified complementary strand compared to the modified strand in the duplex is ∼10 times smaller. The formation of tandem lesions is observed even at low levels of irradiation corresponding to “single-hit” conditions when less than ∼10% of the oligonucleotide strands are damaged. A plausible mechanism for this observation is discussed