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

Abstract

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.

Figure 1

Kinetics of decay of BPT^•+ radicals recorded at 455 nm in solution in the absence of DNA. Panel A: transient absorption spectra of BPT^•+ radicals and hydrated electrons (e⁻) after a single-laser pulse excitation. Panels B and C: decay profiles of BPT^•+ radicals shown on two different time scales. The experiments were recorded after photoexcitation of free BPT (8.3 μM) by actinic 355 nm Nd: Yag laser pulses in air-equilibrated buffer solutions (pH 7.5) containing 100 mM NaCl.

Figure 2

Decay profiles of BPT^•+ radicals recorded at 455 nm in the presence the oligonucleotide duplex 3d (100 μM) that contains two contiguous guanines (…GG… sequence context). Panel A: transient absorptions recorded in air-equilibrated (black) and N₂O-purged (red) solutions; the inset shows the decay of hydrated electrons at 650 nm. Panel B: complete decay of BPT^•+ in N₂O-purged solution. The kinetic traces were recorded after photoexcitation with actinic 355 nm Nd: Yag laser pulses of BPT (8.3 μM) in buffer solutions (pH 7.5) containing 100 mM NaCl.

Figure 3

Rate constants of decay of BPT^•+ radicals (defined by eq. 3) in the absence and presence of different oligonucleotide duplexes (100 μM). Panel A: decay parameters k₁ and k₂. Panel B: amplitudes a₁ and a₂. BPT: no DNA, duplex 1d: two single G near the ends of the duplex; 2d: two single G in the interior of the duplex; 3d: two contiguous G; 4d: three single G in the interior of the duplex; 5d: three contiguous G in the interior of the duplex; 6d: no guanines in the duplex. The values of the kinetic parameters were obtained by the best least squares fits of eq 12 to the BPT^•+ radical decay profiles (455 nm) recorded in air-equilibrated buffer solutions (pH 7.5) containing 100 mM NaCl.

Figure 4

Dependence of the averaged BPT^•+ radical decay constant k_a (defined by eq 14) on the concentration of duplex 4d (three isolated guanines) and 5d (three contiguous guanines). The k_a values were obtained from the rate constants k₁ and k₂ (and a₁, a₂) by a least squares fit of eq 13 to the measured BPT^•+ (455 nm) decay profiles recorded in air-equilibrated buffer solutions (pH 7.5) containing 100 mM NaCl, and different concentrations of the DNA duplexes.

Figure 5

The net rate constants of guanine oxidation (k_G) in DNA duplexes by BPT^•+ radicals (Panel A) and bimolecular rate constants of DNA oxidation by CO₃^•− radicals (Panel B). The rate constants were obtained from the analysis of the BPT^•+ and CO₃^•− decay profiles in the presence of the DNA duplexes containing two separated guanines near the ends of the duplex (1d), two single or isolated guanines in the interior of the duplex (2d), two interior contiguous guanines (duplex 3d), three interior separated guanines (duplex 4d), and three contiguous guanines (duplex 5d). All DNA concentrations were 100 μM in 100 mM NaCl, pH 7.5 buffer solution. Note, that dimensions of k_G and k₉ are different; k_G is a first order rate constant (s⁻¹), and k₉ is a second order rate constant (M⁻¹s⁻¹).

Figure 6

Kinetics of CO₃^•− decay in the absence and presence of an oligonucleotide duplex 5d containing a single contiguous GGG-triplet, recorded after a single-pulse photoexcitation pulse with an actinic 308 nm excimer laser pulse in air-equilibrated buffer solutions (pH 7.5). The inset shows the transient absorption spectrum of CO₃^•− radicals recorded 0.1 ms after the 308 nm laser pulse.

Figure 7

Comparisons of strand cleavage patterns of double-stranded DNA (duplex 7d, 10 μM) generated by CO₃^•− and BPT^•+ radicals, and by riboflavin after incubation with hot piperidine and gel electrophoresis. Autoradiographs of denaturating gels (7 M urea, 20% polyacrylamide gel) showing the cleavage patterns of the duplex 7d labeled at the 5′-termini and excited (A) by a train of 308 nm (~60 mJ/pulse/cm², 10 pulse/s), or (B) by 355 nm laser pulses (20 mJ/pulse/cm², 10 pulse/s), or (C) a 100 W Xe arc lamp in air-equilibrated buffer solutions (pH 7.5). Panel A: Lane G: guanine Maxam-Gilbert sequencing lane of unirradiated sequence; Lane 1: Unirradiated sequence (without piperidine treatment); Lane 2: Unirradiated sequence (after hot piperidine treatment); Lane 3: Unirradiated sequence in the presence of Na₂S₂O₈ (after hot piperidine treatment); Lanes 4 – 11: Irradiated sequence (after hot piperidine treatment) irradiated for 2, 5, 10, 15, 20, 30, 40 and 60 s. Panel B – Lane 1: Unirradiated sequence in the absence of BPT (without piperidine treatment); Lane 2: Unirradiated sequence in the absence of BPT (after hot piperidine treatment); Lane 3: Unirradiated sequence in the presence of BPT (after hot piperidine treatment); Lanes 4 – 9: Irradiated sequence (after hot piperidine treatment) irradiated for 5, 10, 15, 20, 40 and 60 s. Panel C – Lane 1: Unirradiated sequence (without piperidine treatment) in the absence of riboflavin; Lane 2: Unirradiated sequence in the absence of riboflavin (after hot piperidine treatment); Lane 3: Unirradiated sequence in the presence of riboflavin (after hot piperidine treatment) Lanes 4 – 11: Irradiated sequence with riboflavin (after hot piperidine treatment) irradiated for 2, 5, 10, 15, 20, 30, 40 and 60 s.

Figure 8

Histograms of the autoradiographs of denaturating gels shown in Figure 7 for the three oxidants (A) by CO₃^•−, (B) BPT^•+, and (C) riboflavin. In each case, the profiles are derived from the respective lanes for a 15 s irradiation time of duplex 7d.

Figure 9

Histograms of the autoradiographs of denaturating gels (7M urea, 20 % polyacrylamide gel) showing the cleavage patterns generated by BPT^•+ radicals after incubation with hot piperidine in duplexes containing two isolated guanines (duplex 2d, A), a GG doublet (duplex 3d, B), three isolated guanines (duplex 4d, C), and a GGG triplet (duplex 5d, D). The samples were excited by 355 nm laser pulses (20 mJ/pulse/cm², 10 pulse/s) for 20 s.

Figure 10

Oxidative DNA damage triggered by one-electron oxidation of guanine base by BPT^•+ (red arrows) and CO₃^•− (blue arrows) radicals.