Guanine Modifications Following Ionization of DNA Occurs

Publication Date (Web): May 10, 2001. Copyright © 2001 .... I. V. Kurnikov, G. S. M. Tong, M. Madrid, and D. N. Beratan. The Journal of Physical Chem...
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J. Phys. Chem. B 2001, 105, 5283-5290

5283

Guanine Modifications Following Ionization of DNA Occurs Predominantly via Intra- and Not Interstrand Charge Migration: An Experimental and Theoretical Study Peter O’Neill,*,† Anthony W. Parker,‡ Mark A. Plumb,†,§ and Laurens D. A. Siebbeles| MRC Radiation and Genome Stability Unit, Harwell, Oxfordshire OX11 ORD, U.K., Rutherford Appleton Laboratory, Chilton, Oxfordshire OX11 OQX, U.K., Radiation Chemistry Department, IRI, Delft UniVersity of Technology, Mekelweg 15, 2629 JB Delft, The Netherlands ReceiVed: September 27, 2000; In Final Form: January 19, 2001

A series of double-stranded DNA samples of known sequence were used to assess whether 193 nm light induced charge migration in DNA in an aqueous, aerated solution occurs predominantly by inter- or intrastrand processes. Light of 193 nm induces a nonrandom distribution of prompt single strand breaks and base modifications, revealed by Escherichia coli formamido-pyrimidine-DNA glycosylase (Fpg), mainly at guanine with the majority of the DNA sequences. If one strand of the DNA contains a guanine poor region, damage also localizes nonrandomly at adenine, even though a guanine is present within 1-2 base pairs but on the complementary strand. The yield of damage at double guanine (-GG-) sites is greater than at single guanine sites although the specific guanine damage in a -GG- site depends significantly on the local sequence around that site. The experimentally determined distribution of base damage has also been compared with that for distribution of charge density, simulated using a quantum mechanical model assuming charge migrates along either a single strand or either strand of the DNA. In the majority of cases, the distribution of charge density using the model assuming intrastrand charge migration and the distribution of Fpg sensitive sites induced by 193 nm light are predicted. It is proposed that photoionization of DNA results predominantly in sequence dependent intra- and not interstrand charge migration with localization at the most readily oxidized base, generally guanine.

About 50-70% of the lesions induced in cellular DNA by ionizing radiation arise from direct energy deposition events, many of which ionize the DNA.1 Direct ionization of DNA yields electron holes (radical cations) and electron adducts (radical anions).1,2 Since these charges migrate along the DNA strands,1-3 it is thought that certain deoxynucleotides might be “targeted” as sites of damage as a result of migration of the initially formed species. The resulting stable products, if not processed by the cells repair machinery, may contribute to the deleterious effects of ionizing radiation. For instance, 8-oxoguanine is a mutagenic lesion4 and is know to compromise the processing of a vicinal damage.5,6 The first studies using dry or frozen aqueous solutions of DNA showed that ionization of DNA results in charge migration, where the electron hole migrates less than 8 base pairs and localizes to a guanine base.2,7,8 Localization of damage at guanine is consistent with that predicted from the ionization potential values for the deoxynucleotides, where the ease of oxidation follows the trend G > A > C > T.9 In aqueous solution at 293 K, the oxidative path may be simulated using 193 nm light, which directly photoionizes the nucleobases of DNA in monophotonic processes3,10 to give their radical cations. The subsequent migration of the hole to the most easily oxidized base,11 guanine, results in damage localized * E-mail: [email protected]. Tel.: ++44 1235 834393. Fax: ++44 1235 834776 † MRC. ‡ Lasers for Science Facility. § Present address: Department of Genetics, University of Leicester, Leicester LE1 7RH, U.K. | IRI.

predominantly at guanine, as established using DNA damage specific endonuclease probes, namely E.scherichia coli formamidopyrimidine-DNA-glycosylase (Fpg) and E. coli endonuclease III (Nth), which convert oxidized purines or pyrimidines, respectively, into strand breaks.12 The major damage arising from the radical cation of guanine is 8-oxoguanine, which is not particularly alkali sensitive13 but is efficiently excised by Fpg. The yield of photodamage such as pyrimidine dimers and oxidized pyrimidines induced by 193 nm light is relatively minor,14 resulting in “targeted” damage at guanine. Synthetic photooxidants associated with DNA have also been used to initiate oxidation15-21 and guanine identified as the major site of localization of DNA damage following photoinduced charge migration. The mechanism of charge migration at room temperature and the distance dependence of charge migration remain under debate. Several studies indicate charge transfer occurs over a few base pairs; however, recent studies show that long-range photoinduced charge transfer takes place and the efficiency is distance dependent, occurring up to distances of ∼20 nm.18,20,21 This transfer may be modulated by intervening sequences such as 5′-TA dinucleotide repeats19,20 but the dynamics of DNA have been proposed to diminish the effects of intervening sequence between the guanine trapping sites.21 Further, a recent study demonstrates that intrastrand charge migration in DNA occurs preferentially22 in contrast to the interstrand migration inferred from other studies.18 Proposed mechanisms of radical cation transfer include delocalization of the radical cation through a continuous molecular orbital involving donor, acceptor and the intertwining base pairs,23-25 multistep incoherent hopping between the redox centers by thermal activation,23-25 and the formation of a self-trapping

10.1021/jp003514t CCC: $20.00 © 2001 American Chemical Society Published on Web 05/10/2001

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TABLE 1: DNA Strands Containing Specific Sequences. the Site for Normalization of the Yields for Sequences 2-6 Are Highlighted 1) sequence A7gA7 5′-..CGGTTGGAGTCAGCGCAGATCTGCCGGTTAAAAAAAGAAAAAAATTCGCCGC.. 2) sequence for A7CgCA7 5′-..CGCATCGCTTCGCTTAAAAAAACGCAAAAAAATTCGCATGGAATTCGATAT.. 3) sequence T7gCgT7 3′-..AAGCGAAGCGAATTTTTTTTGCGTTTTTTTAAGCGTACCTAAGCTAT.. 4) sequence A5T2gT2A5 5′-..TCCAGGTCTGTTTGGAGTCTGCAGGTTAAAAATTGTTAAAAATTGGAATTCGATAT.. 5) sequence T5A2CA2T5 3′-..CAGACGTCCAATTTTTAACAATTTTTAACCTTAAGCTAT.. 6) sequence A7CA7 3′-..ACAAACCTCAGAGGTCCAAAAAAACAAAAAAACCTAGGAGCTAT..

polaron that migrates by thermally activated hopping.21 Theoretical approaches to simulate radical cation migration indicate that several mechanisms may be involved, their relative contributions reflecting the specific base sequence between acceptor and donor sites.23-25 We have previously demonstrated electron hole migration to guanine, following photoionization (193 nm) under aerobic conditions of a 32P 5′-end-labeled double-stranded DNA restriction fragment, from the sequence dependent yields of prompt DNA single strand breaks (ssb),3,11 alkali-labile sites,3 and Fpg sensitive sites at oxidized purines.12,14 This strategy3,12 is employed to analyze specific DNA sequences and specifically address the question of inter- and intrastrand charge migration. The experimentally determined distributions of damage have been compared with simulations of charge distributions obtained using a quantum mechanical model, which has been successfully applied earlier to describe charge transfer through donorDNA-acceptor systems.25,26 Experimental Section Preparation of 32P-Labeled Restriction Fragments and Fpg Preparation. Oligonucelotides of known sequence were purchased from Cruachem. Double-stranded oligonucleotides with either SstII 3′ overhangs, or BamHI and EcoRI 5′ overhangs were prepared by hybridization of complementary singlestranded oligonucleotides and cloned into the bacterial pSKII plasmid vector. Plasmid DNA was digested with BssHII and 5′ end-labeled with [γ-32P]-ATP.3,12 After a secondary digestion with XhoI or SstI, 5′ 32P-end-labeled double-stranded restriction fragments were resolved by low melting point agarose gel electrophoresis and purified using a Qiagen gel extraction kit. The specific DNA sequences are shown in Table 1. Purified Proteins. Purified Fpg protein was a generous gift from Dr. Roldan-Arjona (ICRF).27 Exposure of DNA to 193 nm Light and Sequence Analyze. The 32P-labeled DNA fragment, premixed with yeast tRNA (∼0.5 µmol dm-3) (A193 ) 1) and dissolved in 1 mmol dm-3 sodium perchlorate at pH 7.0, was photolyzed under aerobic conditions with193 nm light (Lambda Physik LPX210i excimer laser, ArF) in a cuvette of 1 mm path length, as described previously in detail.12 After irradiation, the sample was separated into fractions and precipitated with ethanol and then probed for (i) prompt ssb, (ii) hot alkali sensitive lesions (1 mol dm-3 piperidine and incubated at 90 °C (30 min), followed by repeated lyophilizations in water), and (iii) Fpg sensitive lesions (30 ng of protein/µg of DNA/RNA, buffer ) 0.5 mmol dm-3 dithiothreitol, 0.2 mg/mL BSA, 0.1 mol dm-3 KCl, 0.5 mmol dm-3 EDTA, 40 mmol dm-3 Hepes-KOH, pH 8, 37 °C, 1 h). After digestion, samples iii were mixed with EDTA (final concentration 100 mmol dm-3), precipitated with ethanol, and washed with 70% (v/v) ethanol. Maxam and Gilbert chemical sequenc-

ing reactions were as described.28 Heat-denatured DNA samples (95% formamide, 90 °C, 1 min) were resolved by electrophoresis (6% polyacrylamide/50% urea (wt/vol) gel) and observed by autoradiography. Autoradiograms were quantified for strand breakage using a Fujitron CCD video camera fitted to a Fotodyne camera controller and a Macintosh processor (Collage version 2, Image Dynamics Corp., 1993). Oligonucleotides 2-6 contain a 5′-CGA sequence, as shown in Table 1. Therefore, within a given oligonucleotide the relative extent of strand breakage at a given site may be normalized to that at the guanine within the indicated 5′-CGA sequence. This normalization then allows a comparison of the extent of strand breakage within an oligonucleotide and at the various sites between oligonucleotides 2-6. The experiments were repeated twice per oligonucleotide for a given photon fluence and it is estimated that the errors in the relative yields of damage are e10% after normalization of each experiment, as described above. Since sequence 1 does not contain a comparable 5′-CGA site, only internal comparisons of the extent of strand breakage at the various sites have been made. The error from replicate experiments with sequence 1 was determined to be 8%. The sequence dependence of hydroxyl radical induced strand breakage was determined by 60Co γ-irradiation of the 32P-labeled DNA fragments, dissolved in 1 mmol dm-3 sodium perchlorate at pH 7.6, with a dose of 30-70 Gy. The irradiated samples were analyzed for strand breakage as described above. Computer Simulations. To study theoretically the charge distribution of an excess hole produced on DNA by photoionization with 193 nm light, we used a model that combines a quantum mechanical description for the hole on the DNA chain with a simple description of dynamic fluctuations in the DNA chain.25,26,29 The model includes both diffusive charge transport between sites with similar energy and tunneling transport through sequences of sites with higher energy. We have considered both charge transport along a single strand (intrastrand) and transport through either strand (interstrand) of the DNA. The charge was described using the tight-binding method. The wave function of the charge is a time-dependent superposition of states |n〉 located on different sites:

|Ψ(t)〉 )

∑n cn(t)|n〉

(1)

In the simulations of intrastrand transport the sites correspond to the purine and pyrimidine bases (i.e., G, A, C, and T), while in the case of interstrand transport the sites correspond to the GC and AT base pairs. The charge density on the nth site at time t is given by

Qn(t) ) |cn(t)|2

(2)

Guanine Modifications Following Ionization of DNA

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The tight-binding Hamiltonian that determines the evolution of the wave function is given by

Hq )

+ an + a+ ∑n [na+n an - b(an+1 n an+1)]

(3)

where a+ n and an are the creation and annihilation operators for a charge at the nth site, respectively, b is the transfer integral (electronic coupling between neighboring sites), and n is the energy of the charge when it is localized at the nth site. The site energy consists of a static term and a time-dependent term, i.e., n ) n,stat + n,dyn(t). The time-dependent term represents dynamic fluctuations in the DNA, which causes dephasing and provides a heat bath for the charge carrier, as discussed below. For the description of a hole on a DNA strand the static term n,stat corresponds to the ionization potential of a site. The sequence of the base pairs in the DNA is introduced in the model by taking the static components of the site energies, n,stat, equal to the different ionization potentials of the individual bases for a simulation of intrastrand transport or equal to the ionization potentials of the base pairs for the case of interstrand transport. These ionization potentials were taken from an ab initio study performed by Hutter and Clark.30 The values reported in that work are 8.21 eV for G, 8.54 eV for A, 8.88 eV for C, and 9.16 eV for T, 7.51 eV for a GC base pair, and 8.06 eV for an AT base pair. The value of the charge-transfer integral b will depend on the specific combination of bases on the adjacent sites. Because of the lack of information on the charge-transfer integral for different combinations of bases we have used a single value of b ) 0.11 eV for all combinations of bases; see refs 25 and 26. The dynamic fluctuations in the DNA, which act as a heat bath for the charge, are taken into account by the time-dependent term in the site energies in eq 3. The sites in the DNA chain are considered as coupled harmonic oscillators with mass M and vibration frequency ω. The coupling between the charge and the oscillators is assumed to be linear and accounted for with a proportionality constant g. The effect of dynamic fluctuations can thus be brought into account by the Hamiltonian:

Hv )

{

∑n

P2n(t)

}

1 + Mω2[xn+1(t) - xn(t) - xeq]2 + 2M 2

∑n g[xn+1(t) - xn-1(t) - 2xeq]a+n an

(4)

In this equation pn(t) and xn(t) are the momentum and the position of the nth oscillator at time t, respectively and xeq is the equilibrium distance between adjacent oscillators. The first term in eq 4 describes the harmonic oscillators, while the second term accounts for the coupling between the charge, which is described as a quantum particle and the oscillators that are treated classically. The contribution of the dynamic disorder to the site energy of the hole in eq 3 is thus

n,dyn(t) ) g[xn+1(t) - xn-1(t) - 2xeq]

(5)

The distance between the equilibrium positions of the oscillators was set equal to 3.4 Å, which corresponds to the distance between the base pairs in DNA. The mass of the oscillators, M, the oscillation period, and the coupling constant, g, were taken from the values used previously,25,26 i.e., 60 times the proton mass, 0.45 ps and 0.51 eV/Å, respectively. The initial velocities of the oscillators were sampled from a Boltzmann distribution

at a temperature of 293 K. The initial positions of the oscillators were taken as their equilibrium positions. The velocities and positions were propagated in time until the system of coupled oscillators had reached equilibrium. After this the charge was introduced on the DNA sequence. It was assumed that photoionization of DNA leads to the production of a hole that is initially localized on a base (simulation of intrastrand transport) or on a base pair (simulation of interstrand transport). Hence, if the index of the initial site is i, the initial conditions for the coefficients in eq 1 are cn)i(t)0) ) 1 and cn*i(t)0) ) 0. The wave function and the oscillators were propagated in time by applying a timedependent self-consistent-field formalism31 with the total Hamiltonian, Htot, equal to the sum of eqs 3 and 4, Htot ) Hq + Hv. According to this formalism, the wave function is propagated during a time step dt that is small enough that the positions of the harmonic oscillators can, to a good approximation, be considered fixed. The coefficients cn(t) were obtained numerically by integration of the first-order differential equations that follow from substituting the wave function in eq 1 into the timedependent Schro¨dinger equation ip∂|Ψ(t)〉/∂t ) H|Ψ(t)〉. To propagate the positions and velocities of the oscillators, the total Hamiltonian is averaged over the wave function of the charge given in eq 1. This yields the classical vibrational Hamiltonian from which the first-order differential equations for the velocities and positions of the oscillators can be obtained. These equations were integrated numerically to obtain the velocities and positions after a time step dt, during which the wave function of eq 1 is considered constant. This procedure is repeated until a preset time limit is reached. For each initial site of the hole a simulation of the evolution of the wave function was performed. To compare the simulated results with those from the experiments, the weighted average of the resulting charge distributions was calculated with weights equal to the probability that a hole is produced at the initial site. This probability was assumed to be proportional to the product of the extinction coefficient, A, and photoionization quantum yield, φ, for 193 nm light. For the simulations of intrastrand hole transport the following values10 were used: A ) 25900 M-1 cm-1 and φ ) 0.044 for G, A ) 18600 M-1 cm-1 and φ ) 0.033 for A, A ) 19500 M-1 cm-1 and φ ) 0.029 for C, and A ) 5700 M-1 cm-1 and φ ) 0.055 for T. In the simulations of interstrand charge transport the extinction coefficients and quantum yields of the base pairs were taken equal to the average of those for the single bases. For each initial site of the hole, the charge distribution was averaged over 6 realizations of the initial velocities of the harmonic oscillators, which was found to be sufficient for the results to be independent of the number of realizations. Since the DNA consist of typically 50 base pairs containing the cloned sequence, the average charge distribution was obtained from typically 300 simulations. Results With all the DNA sequences studied, the induction of prompt ssb by hydroxyl radicals is essentially random (see Figures 2, lane 4), as there is little effect of DNA sequence. Photoionization also induces a low sequence-independent yield of prompt ssbs (see Figure 2a, lane 3 for example), assumed to arise from random ionization of the phosphate backbone.3,32 Fpg protein, which converts oxidized guanine into ssb, was used to convert those sites, which are not that sensitive13 to hot piperidine treatment, into ssbs. Charge migration was assessed for 193 nm light induced prompt ssb and Fpg sensitive sites using a “guanine-poor” DNA

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Figure 1. (a) Base specificity of Fpg excised damage (lane 2, energy 5 mJ) of the DNA 191 bp fragment (A7GA7) irradiated under aerobic conditions with 193 nm light. Lane 1 shows Maxam Gilbert G+A sequencing markers. The distribution of the yield of Fpg detected damage (b) and charge density (c) on sequence.

sequence, e.g. A7GA7. Ssb and Fpg sensitive sites are produced predominantly at guanine (Figure 1a, lane 2) as previously shown with random DNA sequences.12 The extent of Fpg sensitive damage at guanine in the A7GA7 region was compared with the extent of guanine damage in nonadenine rich regions. Although photoionization of adenines occurs within this A7GA7 region, damage at these adenine residues was not observed; see Figure 1a. Further, significant damage above background was not observed at the other nucleobases, verified12 using endonuclease III. However, the extent of guanine damage, revealed by Fpg, in the adenine-rich environment (A7GA7, (I)) is ∼9x greater, from comparison of band intensities, than that for guanine damage at AGA (I′), within the nonadenine rich sequence and other single guanine sites, as shown in Figure 1b. It is inferred that the adenine radical cations formed within A7GA7 predominantly lead to enhanced damage at guanine (I). The extent of guanine damage in the CGC and CGGT sequences flanking the A7GA7 region is 10 ( 2% and 55 ( 6% of that at respective CGC and CGGT sequences outside the adenine rich sequence. As previously shown,3,12 the extent of 193 nm light induced guanine oxidation at GG sites is greater than that at single G sites, reflecting their different redox potentials.33,34 However, the extent of oxidation of a single guanine at site I is similar to that at the GG sites shown in Figure 1b, consistent with enhancement of damage at G in site I due to migration of charge from the neighboring adenines. Comparison of the extent of damage at both guanines in a GG site is very dependent on the flanking base moieties as shown in Table 2. For instance, guanine cleavage predominantly occurs at the 5′-G in 5′-CGGT compared with similar levels of cleavage at both guanines in 5′-TGGA sequence. Corresponding cleavage patterns were induced by photooxidants.21,35 The simulated distribution of charge density in the A7GA7 sequence is shown in Figure 1c based on the theoretical model described above25 and assuming the charge migrates along a single strand (intrastrand). To avoid chain end effects, typically 5-10 bases adjacent to both ends of the sequences shown in the graphs were included in the simulations, as discussed below.

TABLE 2: Sites of Damage Induced by 193 nm Light within Double Guanine Sequences As Revealed by Fpg Protein Treatment sequence

relative damage in 5′-GG-sites 5′-G:3′G

ref

5 ′-TGGA 5′-AGGT 5′-TGGC 5′-AGGA 5′-CGGT 5′-TGGT

∼50:50 >80% >80% ∼50:50 >80% >80%

this study, 3, 35 this study, 21 this study, 3 this study 3, 21, 33 3, 34

The distribution of charge was found not to change significantly at t > 1.5 ns. When it was assumed that charge may be transported by either strand (interstrand, data not shown), the simulated distributions of charge density do not provide such good comparisons with the experimental distributions of Fpg sensitive sites as that based on the intrastrand model. In comparing the simulated distribution of charge density with the experimental distribution of Fpg sensitive sites, it has been assumed that the conversion of damage sites recognized by Fpg (e.g., oxidized guanine) into ssbs is quantitative and that the charge density at a specific site is proportional to the yield of damage. Since the majority of the sites with enhanced charge density are at guanines, it is assumed that the rate of conversion of the guanine radical cation into stable products is independent of the local sequence around the guanine. Low levels of charge are distributed at all nucleotide sites (see above) but significantly enhanced levels of charge density are located at guanine sites. The distributions of charge density at guanine sites qualitatively agree with the distribution of Fpg sensitive sites, derived experimentally, with the following exceptions. In 5′-CGGT sequence (see Figure 1c) both guanine residues carry significant charge density but the 5′-G is predominantly excised, as shown in Table 2. In the 5′-TGGAGT, the highlighted guanine carries charge from the simulations but is not excised by Fpg. One possible explanation is that the charge-transfer integral b in eq 3 is different for different combinations of bases. This affects both the rate of charge migration between the bases and the effective redox potentials of the different guanines in -GG-

Guanine Modifications Following Ionization of DNA

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Figure 2. (a) Base specificity of frank ssb (lane 3, energy 5 mJ) and Fpg excised damage (lanes 1, energy 2.5 mJ) and in the DNA (A7CGCA7) irradiated under aerobic conditions with 193 nm light. Lane 2 shows Maxam Gilbert G+A sequencing markers and lane 4 hydroxyl radical cleavage sites. The distribution of the yield of Fpg detected damage (b) and charge density (c) on sequence.

SCHEME 1. Relative Extent of Fpg Excised Damage Induced by 193 nm Light within the Following Complementary Strands of DNAa

a

Figure 3. Yield of Fpg detected damage (a) and the distribution of charge density (b) on sequence in the DNA (T7CGCT7) irradiated under aerobic conditions (2.5 mJ) with 193 nm light.

sequences33,34 and may result in redistribution of the charge in multi-G sites. Since damage is enhanced at guanine with A7GA7, the simulated distributions of charge density and of Fpg sensitive sites were determined on both strands of a double-stranded oligonucleotide containing the A7CGCA7 (Figure 2) and T7GCGT7 (Figure 3) sequences, to assess the effect of less readily oxidized pyrimidine nucleobases. 193 nm light induces ssb (Figure 2a lane 3) and Fpg sensitive sites at guanines as shown in Figures 2a (lane 1), 2b, and 3a. Piperidine treatment only causes a small increase (less than a factor of 2) in the yield of ssb above that of prompt ssb.14 The yield of piperidine sensitive sites is at least an order of magnitude less than that revealed by Fpg.14 With the A7CGCA7 sequence, where cytosines have been inserted between guanine and adenine (Figure 2b), the extent of Fpg detected damage induced at the guanine (I) is only 2.3× greater than that at the equivalent guanine (I′), significantly less (∼4×) than that seen with A7GA7. The simulated distribution of charge density (Figure 2c) is qualitatively similar to that for the Fpg sensitive sites in Figure 2b. With the complementary T7GCGT7 sequence, Fpg sensitive sites induced by 193 nm light

See text for calibration between oligonucleotides.

and charge density are shown in Figure 3a,b, respectively. The amount of charge density and damage at the guanines within the sequence T7GCGT7 are significantly less than that at guanines within the rest of the sequence. Since T7GCGT7 and A7CGCA7 are complementary, the relative yields of excision of damage by Fpg at guanine sites are shown in Scheme 1, following normalization to the respective yield of damage at guanine in the 5′-CGA sites (see Table 1 and the Experimental Section). The extent of excision of Fpg detected damage is significantly reduced at the guanine within the T7CGCT7 sequence and enhanced at the guanine in the A7CGCA7 sequence, compared with the extent of excision of damage at the other equivalent sites. The simulated charge densities show the same effect; see Figures 2c and 3b. Insertion of less readily oxidized cytosine between adenine and guanine in A7CGCA7 (cf. A7GA7, Figure 1) significantly reduces enhancement of charge density and Fpg detected damage at the guanine within A7CGCA7, consistent with the inhibitory effect of charge migration in single-stranded oligonucleotides by pyrimidines.36 This effect of pyrimidines is more evident from the reduced amount of charge density and Fpg detected damage at the guanine in T7GCGT7 (see Scheme 1). Since it is inferred that pyrimidines may reduce 193 nm light induced charge migration in DNA, the distributions of charge density and Fpg sensitive sites were determined in the comple-

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Figure 4. Distribution of the yield of Fpg detected damage ((a) and (c)) and charge density ((b) and (d)) on sequence in the DNA (A5T2GT2A5) ((a) and (b)) and T5A2CA2T5 ((c) and (d)) irradiated under aerobic conditions (2.5 mJ) with 193 nm light.

mentary A5T2GT2A5 and T5A2CA2T5 sequences. Furthermore, in the guanine poor T5A2CA2T sequence, it is possible that charge density and damage induced by 193 nm light may become localized at adenine, as the next more readily oxidized base after guanine.9 The distribution of Fpg sensitive sites in the DNA containing A5T2GT2A5 and T5A2CA5T5 are shown in Figure 4a,c. The Fpg sensitive sites are localized at guanine and the relative yields are shown in Scheme 1, obtained by normalization, as described in the Experimental Section. Several points arise from the distribution of Fpg sensitive sites induced in these DNA sequences by 193 nm light. First, the extent of Fpg sensitive sites at guanine (I) situated in the specific sequence A5T2GT2A5 is ∼0.8 of that at a comparable flanking guanine site (I′) (Figure 4a), consistent with the inserted thymines having an inhibitory effect on migration of charge from the adenines to this guanine. Second, Fpg sensitive damage at adenine (Figure 4c) and enhanced charge density at adenine (Figure 4d) are present in the strand containing T5A5CA5T5 even though guanines are present within 1-2 base pair(s) on the complementary strand (see Scheme 1). The extent of damage at the adenines is comparable with that at the central guanine in the A5T2GT2A5 sequence and significantly greater than that seen for prompt ssb (data not shown). Third, the excision at both guanines is significant in the 5′ TGGA sequence (Figure 4a) and consistent with the cleavage pattern at the 5′-TGGA site seen in Figure 2b. Only the 5′ guanine is excised in the 5′AGGT sequence (Figure 4a), consistent with previous specificity, as shown in Table 2. Further, the highlighted guanine in the 5′-TGGAGT sequence is not excised, consistent with the

observation in the equivalent sequence shown in Figure 1b. It was inferred above that the damage is transferred from 3′-G to the 5′-G. Since enhanced charge density and Fpg sensitive sites are present at specific adenines in the guanine deficient T5A2CA2T5 sequence (Figure 4c,d), the distributions of charge density and Fpg sensitive sites induced by 193 nm light were determined in a A7CA7 sequence and are shown in Figure 5. Although Fpg sensitive sites are produced at guanine within this fragment (Figure 5a), Fpg sensitive sites are produced at certain adenines within the A7CA7 sequence. Even in the simulated distribution of charge density (Figure 5b), slightly elevated densities are seen at the adenine moieties within the A7CA7 sequence, although not restricted to the specific adenines observed experimentally for Fpg sites. The Fpg specific sites detected in -GG- sites are shown in Table 2. Discussion From simulations of 193 nm light induced charge migration in DNA, the main charge density is localized at guanines (oligonucleotides 1-4 in Table 1). The main site of persistent DNA damage determined experimentally from damage excision by Fpg is at guanine, consistent with previous observations.12 The distribution of charge density and of Fpg sensitive sites are similar but with a few minor differences. If DNA contains a guanine poor region, as in strands 5 and 6 (Table 1), then 193 nm induced damage may localize at adenine, consistent with the enhanced simulated charge density seen at adenines, especially in sequence 5.

Guanine Modifications Following Ionization of DNA

Figure 5. Yield of Fpg detected damage (a) and the distribution of charge density (b) on sequence in the DNA (A7CA7) irradiated under aerobic conditions (2.5 mJ) with 193 nm light.

On the basis of the extinction coefficients and quantum yields of photoionization of the individual nucleobases, 193 nm induced photoionization of DNA occurs at any of the nucleobases monophotonically3 but with different probabilities. The simulated charge density becomes localized, predominantly at guanine, within 1.5 ns, compatible with the one-electron oxidized radical of guanine being the main intermediate at very short times.3,11 At much longer times following the calculated times for the charge density distribution to reach equilibrium, the one electron guanine radical is known to undergo irreversible hydration37 within about11 0.05 s to yield ultimately 8-oxoguanine, a substrate for Fpg. If it is assumed that the rate of hydration of the guanine radical is independent of local sequence and irreversible hydration is the major process leading to persistent damage, then the distribution of hydrated guanine radicals should be similar to that of the charge density, since the latter would not be perturbed by irreversible hydration processes. The dependence of the yield of Fpg sensitive sites on photon fluence14 is indeed linear. However, a potential complication is the buildup of oxidation products of guanine, namely 8-oxoguanine, which becomes the preferred sink for charge localization.38,39 The products of oxidation of 8-oxoguanine are exquisitely alkali labile.40 The yield of alkali labile sites is low,14 so that complications from oxidation of the 8-oxoguanine produced is minimal. It is essential to demonstrate in studies of guanine oxidation in DNA that the yields of guanine damage are linearly dependent upon the photon fluence. Further, for a given photon fluence, the extent of damage at a particular guanine site revealed by hot piperidine treatment is minor14 compared with that after treatment with Fpg. From the simulations of the distribution of charge density, it is inferred that the favored route for charge migration is through intrastrand processes with enhancement of damage at guanine. The simulated charge distributions for intrastrand charge migration are in qualitative agreement with our experimental results, while simulations of interstrand charge migration do not agree with our experimental findings. For instance, the simulated charge distribution due to intrastrand migration in A7CGCA7 agrees well with that experimentally determined for Fpg sensitive sites, whereas simulations of interstrand migration predict similar amounts of charge density on guanine and cytosine sites. That the agreement between experimental data

J. Phys. Chem. B, Vol. 105, No. 22, 2001 5289 and simulations is not quantitative may be due to the fact that the experimental sensitivity is determined not only by the charge density but also by the local DNA sequence and the processes leading to conversion of the radical cations into substrates for Fpg. In the simulations, the value of the transfer integral b in eq 3 was taken to be the same for all combinations of bases, due to the lack of information on the values for different combinations of base pairs. The use of different b values leads to different charge-transfer rates between the bases and to different energies of the molecular states that result from mixing of the states localized on the bases (states |n〉 in eq 1). However, if in the simulations the transfer integral between adenine and thymine were smaller, the tunneling of the charge from the adenine regions through the thymine regions to the guanine would become less likely. Charge migration involving hopping/ tunneling may also compete with proton transfer to their complementary bases.41 Since significant yields of adenine damage (Scheme 1 and Figure 5a) were detected in guanine-deficient regions of DNA even though a guanine is present on the complementary strand within 1-2 base pairs, migration of charge with localization at adenine/guanine is also consistent with charge transfer occurring mainly via intrastrand processes in DNA. The rate of irreversible transformation of the one electron oxidized adenine would have to compete favorably with that for interstrand charge transfer to a neighboring guanine. Indeed, cleavage at adenine is inhibited if guanine is within a few bases on the same strand; e.g., compare A7CA7 and A7GA7. Since the induction of ssb by OH radicals in these sequences is random, any sequence modifications to the secondary/tertiary structure of DNA is minor. These observations provide strong evidence that interstrand charge transfer to guanine is not the main route for charge migration. This proposal is reinforced by the presence of adenine damage in T5A2CA2T5. The 3-4 pyrimidines separating the adenine residues from the nearest flanking guanine within its own strand suggests, at least, a partial barrier for charge migration. This evidence is consistent with reduced enhancement of damage at the central guanine in the A5T2GT2A5 and A7CGCA7 sequences (Scheme 1) and the influence of A/T sites on the distance dependence for photooxidant induced charge migration.15,18,19 This effect is not reproduced by the simulations of the charge density (cf. Figure 4b), possibly due to the integral (b in eq 3) for charge transfer between adenine and thymine being smaller than the value used in the calculations. Giesse et al.42 demonstrated that the precursor sugar radical cation initially oxidizes the nearest guanine by an interstrand-transfer process followed by charge migration to guanines present on the same strand. Subsequently, it was inferred18 that the sugar radical cation initially oxidizes the neighboring guanine on the same strand. This oxidized guanine then undergoes interstrand oxidation of a guanine, which is one base pair away on the complementary strand. It should be noted that the nearest neighbor to the proposed, initially oxidized guanine is greater than 16 base pairs away. Therefore, Giesse’s studies18 do not argue against intrastrand charge migration between the nucleobases since the sugar radical cation may initiate oxidation of guanine on the complementary strand.42 It was suggested that variations in the value of β for photoinduced charge migration in DNA in water at room temperature reflect differences in the probability of intrastrand and interstrand charge migration.22 As previously shown by 193 nm light3,12 and photooxidants,15-20,33-35 damage at -GG- sites is significantly greater than at single guanine sites, presumably reflecting the lower ionization potentials of -GG- sites.33,34 The distribution

5290 J. Phys. Chem. B, Vol. 105, No. 22, 2001 of oxidative damage at -GG- sites, shown in Table 2, depends significantly on the local sequence around the -GG- sites. Whereas with 5′-TGGA and 5′-AGGA, both guanines may be damaged, with the other sequences showing damage occurs preferentially at the 5′-guanine. In the simulations, the effects of the local sequence may be reproduced if differences in the values of the charge-transfer integrals between different combinations of bases could be taken into account. The values of the charge-transfer integrals not only determine the chargetransfer rates but also affect the available energy levels for the hole due to different mixing of the orbitals on the bases. The latter depends on the local sequence of bases and consequently oxidation of a -GG- site might occur mainly at the 5′-guanine. In conclusion, photoionization of DNA results predominantly in a sequence dependent intra- and not interstrand charge migration of the radical cation of the nucleobases to the most easily oxidized base, generally guanine. Acknowledgment. This study was partially funded by the CEC (Contract No. FIGH-CT1999-00005). We thank Dr T. Roldan-Arjona for providing purified proteins. References and Notes (1) O’Neill, P.; Fielden, E. M. AdV. Radiat. Biol. 1993, 17, 53-120. (2) Becker, D.; Sevilla, M. D. AdV. Radiat. Biol. 1993, 17, 121-180. (3) Melvin, T.; Plumb, M. A.; Botchway, S. W.; O’Neill, P.; Parker, A. W. Photochem. Photobiol. 1995, 61, 584-591. (4) Friedberg, E. C.; Walker, G. C.; Siede, W. DNA Repair and Mutagenesis; ASM Press: Washington, DC, 1995 (and references therein). (5) Harrison, L.; Hatahet, Z.; Wallace, S. S. J. Mol. Biol., 1999, 290, 667-684. (6) David-Cordonnier, M.-D.; Lavel, J.; O’Neill, P. J. Biol. Chem., 2000, 275, 11865-11873. (7) Grasslund, A.; Ehrenberg, A.; Rupprecht, A.; Stro¨m, G. Biochem. Biophys. Acta 1971, 254, 172-86. (8) Debije, M. G.; Milano, M. T.; Bernhard, W. A. Angew. Chem., Int. Ed. Engl. 1999, 38, 2752-2756. (9) Steenken, S.; Jovanovic, S. V. J. Am. Chem. Soc. 1997, 119, 617618. (10) Based on average values for the quantum yield of ionization and extinction coefficients at 193 nm for the mononucleosides. Candeias, L. P.; Steenken, S. J. Am. Chem. Soc. 1992, 114, 699-704. Candeias, L. P.; O’Neill, P.; Jones, G. D. D.; Steenken, S. Int. J. Radiat. Biol. 1992, 61, 15-20. Botchway, S. Ph.D. Thesis, University of Leicester, 1996. (11) Melvin, T.; Botchway, S. W.; Parker, A. W.; O’Neill, P. J. Am. Chem. Soc. 1996, 118, 10031-10036. (12) Melvin, T.; Cunniffe, S. M. T.; O’Neill, P.; Parker, A. W.; RoldanArjona, T. Nucl. Acids Res. 1998, 26, 4935-4942. (13) Cullis, P. M.; Malone, M. E.; Merson-Davies, L. A. J. Am. Chem. Soc. 1996, 118, 2775-2781. (14) Melvin, T.; Cunniffe, S. M. T.; Papworth, D.; Roldan-Arjona, T.; O’Neill, P. Photochem. Photobiol. 1997, 65, 660-665. (15) Brun, A. M.; Harriman, A. J. Am. Chem. Soc. 1992, 114, 36563660. Meade, T. J.; Kayyem, J. F. Angew. Chem., Int. Ed. Engl. 1995, 34,

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