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Nucleic Acids Research Pages 4935-4942  

Guanine is the target for direct ionisation damage in DNA, as detected using excision enzymes
Introduction
Materials And Methods
   Determination of the quantum yield of photoionisation
   Standard protocol for the exposure of DNA to 193 nm light and the subsequent sequence analysis
   Quantification of DNA modifications
   Assay to determine whether 2,2-diaminooxazolone is excised by Nth
   Assay to determine whether 8-oxoGua is excised by the Nth preparation used for these studies
Results
Discussion
Acknowledgements
References

Guanine is the target for direct ionisation damage in DNA, as detected using excision enzymes

Guanine is the target for direct ionisation damage in DNA, as detected using excision enzymes

T. Melvin*, S. M. T. Cunniffe, P. O'Neill, A. W. Parker1 and T. Roldan-Arjona2

MRC Radiation and Genome Stability Unit, Harwell, OX11 ORD, UK, 1Rutherford Appleton Laboratory, Chilton, OX11 OQX, UK and 2Departamento de Genetica, Universidad de Cordoba, 14071-Cordoba, Spain

Received June 30, 1998; Revised and Accepted September 15, 1998

ABSTRACT

Exposure of an aqueous, aerated solution (pH 7) of a double-stranded DNA to 193 nm light, of sufficient energy to ionise DNA, leads to selective, non-random modification at guanine in the form of frank single-strand break (ssb) and base modifications, revealed by treatment with either Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg), Escherichia coli endonuclease III (Nth) or hot piperidine treatment. There is a similar neighbouring base sequence dependence for Fpg- and Nth-sensitive damage as that previously reported for both hot alkali-labile damage and prompt ssb. Low yields of photoproducts, namely pyrimidine dimers, are also revealed using the enzyme T4 endonuclease V (T4 endo V). Although irradiation of DNA with 193 nm light causes photoionisation of all the nucleic acid bases, these results indicate that guanine is the predominant site for localisation of the oxidative damage. These findings are consistent with migration of the radical cation to `target' damage at guanine sites.

INTRODUCTION

Direct ionisation of DNA yields stable products, which contribute to the deleterious effects of ionising radiation (1). The DNA duplex has recently been described as a `[pi]-way' for electrons, between intercalated, highly oxidising/reducing, donor/acceptor pairs (2,3). Since direct ionisation of DNA yields electron holes (radical cations) and electron adducts (radical anions), the charge could likewise migrate along the DNA strand. Indeed, within dry or frozen aqueous solutions of DNA, the electron hole migrates and is localised on the guanine base (4,5). Migration is consistent with that predicted from the ionisation potential values for the deoxynucleotides, where the ease of oxidation follows the trend G > A > C > T (6). Since damage produced by energy deposition in DNA within a cellular environment varies with radiation quality (i.e. [gamma]- or [alpha]-radiation) (7 and references therein), it is proposed that certain deoxynucleotides might be `targeted' as a result of migration of the initially formed species. [gamma]-Irradiation of double-stranded DNA in hydrated films results in enhanced yields of damaged deoxyguanosine residues as a result of migration of the oxidative component of direct ionisation (8).

Generation of direct ionisation events of DNA in an aqueous environment is not easily done using conventional radiolytic methods, since formation of overwhelming yields of water radicals is unavoidable. However, irradiation of aqueous solutions of double-stranded DNA with 193 nm light results in monophotonic ionisation of the bases of DNA, although with different efficiencies (9-11), to yield the radical cation and an electron. Hole migration in single-stranded oligonucleotides has been demonstrated using 193 nm laser light (12). Hole migration was suggested to occur within double-stranded DNA samples in view of the similarity of the optical transient absorption spectra for a series of double-stranded DNA samples and poly(G), but gave little indication about distance and sequence dependence of hole migration processes (13). If hole migration to the most easily oxidised base occurs, this should lead predominantly to the formation of damage at guanine sites via the radical cation. Damage identified from formation of the guanine radical cation in DNA includes: (i) frank single-strand breakage (ssb) (14,15); (ii) alkali-labile ssb (13,15); (iii) 8-oxo-7,8-dihydroguanine (8-oxoGua) (16); (iv) in the presence of oxygen, 2,2-diaminooxazolone (17). Recently, 193 nm irradiation of plasmid DNA has been demonstrated to cause damage, which is excised by Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg), an enzyme known to excise oxidised purine damage (18). The present study was done to determine whether damage, caused by exposure of DNA to 193 nm light, is targeted specifically at guanine or whether other nucleic acid bases are damaged by photo-processes or photo-ionisation events. The yields and the base specificity for damage excised by Fpg, Escherichia coli endonuclease III (Nth) and bacteriophage T4 endonuclease V (T4 endo V) were investigated and are reported here. These excision enzymes are known to recognise various types of base damage: oxidised purine, pyrimidine and photodimer damages (19,20 and references therein), which may be induced by ionisation and photo-processes of DNA (16,21-25). Excision enzymes have proved useful tools for the determination of both the yield and the nucleic acid base specificity for various damaging agents (26,27), because the site of nucleic acid base damage is revealed as a gap/ssb. The purpose of this study is to identify whether oxidative nucleic acid base damage occurs at specific `hot spots' in DNA as a result of the `direct' effects of ionising radiation, such as [gamma]-radiation.

MATERIALS AND METHODS

Fpg was isolated from the overproducing Escherichia coli strain JM105/pFPG230 following the purification procedure described by Boiteux et al. (28). Nth was isolated from the overproducing E.coli strain [lambda]N99cI857/pHIT1 as described by Asahara et al. (29). Nth and Fpg were probed for contaminating proteins on an SDS-PAGE gel, Fpg could not be detected in the Nth sample even when the gel was heavily loaded.

Determination of the quantum yield of photoionisation

The laser flash photolysis apparatus has been described in detail (13). The quantum yield of photoionisation of plasmid DNA was determined from the optical absorbance at 650 nm, 0.5 µs after pulse irradiation (193 nm, 30 ns) of an argon saturated, aqueous solution of pUC-18 plasmid DNA containing 1 mmol/dm3 sodium perchlorate and referenced relative to that from a sodium chloride solution. (Both solutions had an absorbance at 193 nm of unity.) The quantum yield of photoionisation of sodium chloride was assumed to be 0.46, the same as that for 185 nm light (30).

Standard protocol for the exposure of DNA to 193 nm light and the subsequent sequence analysis

The c-DNA MIP-1[alpha] restriction fragment was prepared as previously described (13). To purify DNA without the use of a phenol extraction, hence minimising oxidised lesions (31) that could be excised by Fpg, maxi plasmid kits, nucleotide purification columns and gel purification columns (Qiagen) were used. The 32P-labelled DNA fragment, premixed with yeast tRNA (~0.5 µmol/dm3) (A193 = 1) and dissolved in 1 mmol/dm3 sodium perchlorate, was photolysed with 193 nm light (Lambda Physik LPX210i excimer laser, ArF) in a cuvette of 1 mm pathlength. 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/dm3 piperidine, incubation at 90°C for 30 min, followed by lyophilisation with water); (iii) Fpg-sensitive lesions (30 ng protein/µg DNA/RNA, buffer = 0.5 mmol/dm3 dithiothreitol, 0.2 mg/ml BSA, 0.1 mol/dm3 KCl, 0.5 mmol/dm3 EDTA, 40 mmol/dm3 HEPES-KOH, pH 8, 37°C, 1 h), (iv) Nth-sensitive lesions (6 ng protein/µg DNA/RNA, buffer as for Fpg, at 37°C, 30 min); (v) T4 endo V-sensitive lesions (45 ng protein/µg DNA/RNA, buffer = 10 mmol/dm3 Tris, 0.1 mol/dm3 NaCl, 10 mmol/dm3 EDTA, 37°C, 15 min). After digestion, samples (iii), (iv) or (v) were mixed with EDTA (final concentration 100 mmol/dm3), precipitated with ethanol and washed with 70% ethanol. Maxam-Gilbert sequences were obtained using reported protocols (32). Heat-denatured DNA samples (95% formamide, 90°C, 1 min) were resolved by electrophoresis (6% polyacrylamide/50% urea w/v gel) and observed by autoradiography. Samples of DNA treated with osmium tetroxide or 248 nm light were probed with Nth and T4 endo V to confirm the enzyme specificity under the reaction conditions used. Autoradiograms were quantified using a Fujitron CCD video camera fitted to a Fotodyne camera controller and a Macintosh processor (Collage v.2; Image Dynamics Corp.).

Quantification of DNA modifications

Plasmid pUC-18 was freshly isolated, dialysed and diluted with a 1 mmol/dm3 aqueous solution of sodium perchlorate such that the optical absorbance (260 nm) of the solution was 0.5. The sample (100 µl, 25 µg/dm3) at pH 7.6 was irradiated in quartz cuvettes (1 mm pathlength, Suprasil I; Hellma) with pulses of laser light (193 nm for 20 ns) from an ArF excimer laser (Lambda Physik), attenuated with a series of dichroic mirrors. Samples were irradiated with light of pulse intensity ~0.2 mJ/cm2 (~20% of the 193 nm light is absorbed by the DNA solution in the cuvette; 33) determined using a Gentec power meter (ED200; LG Products) at the position of irradiation. Argon saturated samples were prepared by carefully bubbling a fine stream of argon gas through the sample within the cuvette for 15 min, prior to irradiation. After exposure, the aqueous samples of the plasmid DNA were immediately treated as follows.

(i) An aliquot of 15 µl of each sample was stored on ice. (ii) The remainder of the sample was precipitated with ethanol and then redissolved in 50 µl of either Nth or T4 endo V assay buffer. The sample was divided into two portions. (iia) The first sample was treated with Nth (6 ng protein/µg DNA) or T4 endo V (45 ng protein/µg DNA). [The quantity of protein/µg DNA used was determined using a series of 193 nm irradiated plasmid samples and concentrations of Nth or T4 endo V (data not shown), the selected concentration of which was sufficient that a higher concentration did not excise further sensitive sites of the irradiated samples.] (iib) The second sample was diluted with assay buffer only.

Samples (iia) and (iib) were incubated at 37°C for either 30 (Nth) or 15 min (T4 endo V) and subsequently treated with EDTA (pH 8) (final concentration 0.1 mol/dm3), then cooled over ice. Non-irradiated controls were subjected in parallel to the same procedures.

Sample (i), used for the determination of the yield of prompt ssb, was mixed directly with loading buffer containing bromophenol blue. Samples (iia), used for the determination of Nth or T4 endo V active sites caused by exposure to 193 nm, and (iib), used to determine whether there are sites which might be sensitive to the assay conditions, were mixed directly with loading buffer. The various forms of the plasmid DNA were separated overnight at 4°C in the dark on a neutral agarose (0.9%) gel by electrophoresis (Pharmacia GNA200) at a potential of 3 V/cm. Quantification of the DNA forms (closed circular and open circular) was done as previously described (18). The quantum yields of products were calculated using the method of Kochevar et al. (15,34).

Assay to determine whether 2,2-diaminooxazolone is excised by Nth

Plasmid pUC-18 DNA [3 ml, 75 µg/dm3, optical absorbance (260 nm) 1.5, pH 7.6] diluted in an aqueous solution of 1 mmol/dm3 sodium perchlorate was irradiated in quartz cuvettes (10 mm pathlength, Suprasil I; Hellma) with laser light (193 nm for 20 ns). Samples were stirred with a fine stream of oxygen gas. After exposure, the aqueous samples of the plasmid DNA were divided into two equal fractions (i) and (ii) and concentrated using Nanosep 300K filters (Pall Filtron). One fraction (ii) was incubated with Nth (under conditions as described above) and following incubation again filtered using Nanosep filters. Both the residue (iia) and the filtrate, containing any excised lesions (iib), were collected. All of the samples (i), (iia) and (iib) were probed for 2,2-diaminooxazolone by the chromatographic method of Cadet et al. (17,35) (LKB 2152 controller, with two LKB 2150 HPLC pumps, Phenomenex Columbus 5µ C18, 100A, 250 × 4.5 mm column with Waters 420-AC fluorescence detector, 338 nm excitation, 405 nm cut-off filter).

Assay to determine whether 8-oxoGua is excised by the Nth preparation used for these studies

Plasmid DNA was irradiated by an identical procedure as for the assay to determine whether 2,2-diaminooxazolone is excised by Nth (described above). The irradiated sample was incubated with Nth (following conditions described above) and subsequently filtered using Nanosep filters. The filtrate was analysed by HPLC for 8-oxoGua using electrochemical detection (Chromajet electrochemical detector; EDT Instruments Ltd, UK) (36) under conditions which would be sensitive enough to detect excision of 8-oxoGua, if present as 5% of the lesions excised by the Nth. A similarly irradiated plasmid sample which had not been treated with Nth was analysed for the presence of 8-oxoGua using a reported enzymatic digestion and HPLC method (35).

RESULTS

Irradiation of the 32P-end-labelled double-stranded restriction fragment of DNA (250 bp) (MIP-1[alpha] sequence) with 193 nm light results in frank ssb predominantly at the guanine moiety (Fig. 1, lane 2), which is consistent with our previous observations (13). Additionally, cross-linked DNA, in a broad band, migrates to a shorter distance than the undamaged fragment. Treatment of the irradiated fragment with up to a 5-fold concentration excess of Fpg yields an ssb in the duplex at guanine (Fig. 1, lanes 3 and 4), but had no effect on the unirradiated DNA (Fig. 1, lane 6). The electrophoretic mobility of the DNA fragments (Fig. 1, lane 3) following treatment with Fpg is marginally greater than that for the fragments of frank ssb (Fig. 1, lane 2). The relative yields of ssb at each guanine residue following Fpg treatment were determined by image analysis and are shown in Figure 2.


Figure 1. The effect of treatment by Fpg on the base specificity of ssb induction in the DNA fragment of known sequence by 193 nm light. Lane 1 (AG), Maxam-Gilbert (G/A) cleavage products; lane 2 (h[nu]), frank ssb (total fluence = 2 mJ/cm2, A193 nm = 0.1); lane 3 (h[nu] + Fpg), sample treated with Fpg (30 ng/µg DNA); lane 4 [h[nu] + Fpg (5×)] sample treated with Fpg (150 ng/µg DNA); lane 5 (no h[nu]), no light; lane 6 (no h[nu] + Fpg), no light, but treated with Fpg (30 ng/µg DNA).


Figure 2. Relative yields (in arbitrary units) as determined by densitometric analysis of the autoradiograph of Figure 1, lane 3 (h[nu] + Fpg) for ssb at specific sites induced in the DNA fragment of known sequence by 193 nm laser light followed by treatment with Fpg. X, guanine sites where ssb is not detected.

The relative yields of damage excised by Fpg compared with that cleaved by the hot piperidine treatment are very similar (Fig. 3). These histograms show that the distribution of damages or ssb at guanine is non-random with a dependence on neighbouring base sequence which is similar for both Fpg-sensitive sites and hot alkali-labile sites. The absolute quantity of hot alkali-sensitive lesions, compared with that for Fpg-sensitive lesions, is lower; this was determined for a DNA sample irradiated with various total photon fluences and subsequently treated with Fpg or hot alkali. The samples treated with Fpg showed higher levels of damage, as evidenced by the increased shift at the higher total fluence to shorter DNA fragments (data not shown). Additionally, hot alkali treatment of irradiated DNA samples first treated with Fpg does not result in a `significant' increase in damage at the guanine residues compared with similar samples just treated with Fpg. Although the respective yields at each guanine residue have similar neighbouring base sequence effects for the sample treated with Fpg compared with that treated with hot piperidine, there is one adenine residue which is cleaved by the hot piperidine treatment but not by Fpg. This residue is present in a series of adenine residues, flanked by pyrimidine residues (Fig. 4).


Figure 3. Relative yields (in arbitrary units) of damage at guanine induced in DNA by 193 nm light and subsequent treatment with (I) hot piperidine (13) or (II) Fpg (30 ng/µg DNA).


Figure 4. The effect of treatment by Fpg and hot piperidine on the base specificity of ssb induction in the MIP-1[alpha] DNA fragment by 193 nm laser light. Lane 1 (FPG), sample treated with Fpg (30 ng/µg DNA); lane 2 (alkali), sample treated with piperidine (90°C, 30 min); lane 3 (AG), Maxam-Gilbert (G/A) cleavage products.

To determine whether pyrimidine damage is formed by irradiation of DNA with 193 nm light, the sequence specificity for damage detected by the excision enzymes, Nth and T4 endo V, was evaluated. Nth is known to excise oxidised pyrimidine damages, whereas T4 endo V is known to excise pyrimidine photodimers (19). The sequencing gel displayed in Figure 5 was obtained following irradiation of the double-stranded restriction fragment of DNA (250 bp MIP-1[alpha] sequence) with 193 nm light followed by treatment with Fpg, Nth and T4 endo V excision enzymes.

Damage, in DNA irradiated with 193 nm light, is excised by Nth at guanine sites, but not detected at pyrimidine sites (Fig. 5, lane iii). At each Nth excision site two fragments were observed. There is a fragment with an electrophoretic mobility similar to that of the fragment formed by prompt ssb (Fig. 5, lane i) and a fragment with a mobility similar to that formed after Fpg treatment (lane ii) (further details in Discussion). Since guanine damage is excised by Nth, plasmid DNA samples irradiated with 193 nm light were probed for the release of 2,2-diaminooxazolone (or its precursor 2-aminoimadazol-4-one), a known product of the guanine radical cation (17), by this enzyme (assay details in Materials and Methods). Although 2,2-diaminooxazolone is formed by exposure of the DNA to 193 nm light, this base modification is not excised (>95%) by the Nth under the conditions used here and remains intact in the DNA sample. Even though there was no evidence from the SDS-PAGE gel (data not shown) that the Nth sample used was contaminated with Fpg, the ability of this preparation to excise 8-oxoGua was probed. 8-Oxo-7,8-dihydro-deoxyguanosine was shown, by HPLC analysis, to be present in the plasmid DNA sample irradiated with 193 nm light (Materials and Methods). However, there was no detected excision of this lesion, 8-oxoGua, by the Nth preparation under the conditions used. Additionally, the selected concentration of Nth (6 ng protein/µg DNA) was sufficient that a higher concentration did not excise further sensitive sites of the irradiated samples.


Figure 5. The base specificity of prompt and enzyme excision damage induced in the DNA fragment of known sequence by 193 nm light. (i) Frank ssb (total fluence ~7.5 mJ/cm2, A193 nm = 0.1); samples treated with (ii) Fpg (30 ng protein/µg DNA/RNA), (iii) Nth (6 ng protein/µg DNA/RNA), (iv) T4 endo V (45 ng protein/µg DNA/RNA); (v) Maxam-Gilbert (G/A) cleavage products; (vi) Maxam-Gilbert (C) cleavage products. The diagram was obtained from a single gel with the lanes in the order as shown in the photograph, however, for illustrative purposes, the autoradiogram has been photographed at different exposures and the lanes re-aligned with the original, for ease of observation of some of the weaker bands in lanes iii-v.

The sample treated with T4 endo V (Fig. 5, lane iv) contains fragments with a similar electrophoretic mobility as that for the samples in lane i (prompt ssb), consistent with prompt ssb at the guanine site. Also, fragments of lengths consistent with cleavage of damage sites at thymine residues are observed (i.e. Fig. 5, label c; within the sequence GGTTTC, two bands are detected, from two thymine dimer products). There is no detectable excision of damage by T4 endo V within the adenine sequence which contains alkali-labile lesions (Fig. 5, label d, and results above; Fig. 4). There is no detectable cleavage of the unirradiated DNA by Nth and T4 endo V (results not shown).

Figure 6 shows the dependence on fluence of the closed circular DNA fraction of plasmid DNA in an aqueous solution containing 1 mmol/dm3 sodium perchlorate, under oxic and anoxic conditions, irradiated with 193 nm light followed by post-exposure treatment with Nth (Fig. 6a) and T4 endo V (Fig. 6b). The yields of enzyme-induced ssb (Nth or T4 endo V) is linear with fluence. Treatment of similar samples with either the assay buffer only [sample (iib)] or boiled [sample (iiic)] at 37°C results in small differences in the yields of induced ssb as compared with the yield of prompt ssb. The quantum yields of 193 nm light-induced damage excised by the enzymes are shown in Table 1 and have been determined from the D37 values shown in Figure 6 taking into account the dependence for the samples not treated with the enzyme, but treated with the incubation conditions only.

   a

   b

Figure 6. (a) The dependence of the closed circular DNA fraction with total fluence obtained after exposure of plasmid DNA to 193 nm light followed by post-exposure treatment with Nth. [solid square], air saturated samples treated with Nth; [solid circle], air saturated samples incubated in enzyme buffer; [open square], argon saturated samples treated with Nth; [open circle], argon saturated samples incubated in enzyme buffer. (b) The dependence of the closed circular DNA fraction with total fluence obtained after exposure of plasmid DNA to 193 nm light followed by post-exposure treatment with T4 endo V. [solid square], air saturated samples treated with T4 endo V; [solid circle], air saturated samples incubated in enzyme buffer; [open square], argon saturated samples treated with T4 endo V; [open circle], argon saturated samples incubated in enzyme buffer.

Table 1. Quantum yields of damage caused by exposure of pUC-18 plasmid DNA to 193 nm light
Damage/electrons Quantum yields (×10-4)
Air Argon
Photoelectrons - 860 ± 11
Prompt ssb (18) 1.5 ± 0.1 0.9 ± 0.1
Damage excised by Fpg (18) 33.1 ± 3.1 23.8 ± 2.6
Damage excised by Nth 7.3 ± 0.7 6.2 ± 1.3
Damage excised by T4 endo V 5.5 ± 0.6 8.2 ± 0.9

DISCUSSION

Light at 193 nm has been shown to induce photoionisation of all the mononucleosides of DNA (9) and in DNA the resulting radical cations migrate to guanine (12,13). Frank ssb are formed at guanine via a guanine radical cation precursor (14); however, the quantum yield of prompt ssb is very low (15,18). The question arises as to whether the majority of the damage induced by ionisation of DNA by 193 nm light can be ascribed to nucleic acid base modifications. The data presented indicate that the exposure of the DNA to light of this wavelength yields base modifications predominantly at guanine, as revealed by treatment with the excision enzymes, Fpg, Nth and T4 endo V. Firstly, exposure of DNA to 193 nm light induces guanine modifications, which are excised by Fpg to produce a single-strand gap. The yields of guanosine modification, either in the form of frank ssb or damage excised by Fpg, is not equal for each site, but is a reflection of the local DNA sequence. This dependence on the neighbouring base sequence shows similarities to that previously reported for hot alkali-labile damage (Fig. 4; 13). The yield of Fpg-sensitive damage, measured as ssb, is ~20 times that for prompt ssb in plasmid DNA irradiated with 193 nm light (18). Thus much larger yields of guanine modification than frank ssb result from guanine radical cations. The amount of damage of guanine in the form of prompt ssb, hot alkali-labile damage and Fpg excision damage is greatest for neighbouring guanine residues unless this duplex has a 3[prime] thymine residue. Guanine residues in other sequence contexts are damaged to different degrees (Figs 1 and 2). Other groups have reported a neighbouring sequence dependence on the yield of hot alkali-labile guanine modifications formed as a result of oxidative photoinduced electron transfer reactions (37,38). The sequence dependence for the yield of guanine damage follows a trend which is similar to that estimated using ionisation potential values of guanine at various sites within DNA; the ease of oxidation is influenced by molecular orbital interactions between guanine and a neighbourhood base within the DNA helix (39). The fact that the sequence specificity of the yield of damage detected as prompt strand breaks, alkali-labile sites and Fpg-sensitive sites at guanine is similar would lead us to suggest that excision of photoionisation damage by the Fpg is not influenced by the neighbouring base sequence for the damage.

Low yields of damage are excised by Nth from the plasmid DNA irradiated with 193 nm light (Fig. 6a and Table 1). Nth is an enzyme which is known to excise oxidative pyrimidine damage sites (19). Even so, the damage excised by Nth from DNA exposed to 193 nm light under oxic conditions occurs exclusively at the guanine residues (Fig. 5, lane iii). There is no evidence for excision of any oxidative pyrimidine damage (23-25), which would have been excised by Nth if present. The selected concentration of Nth was sufficient that a higher concentration did not excise further sensitive sites, therefore detected excision at guanine sites is not a reflection of relative ease of excision of this damage as compared with possible pyrimidine lesions. It is inferred that migration of the hole from the pyrimidines to the most easily oxidised base, namely guanine (6), occurs more rapidly than the competing pyrimidine radical cation chemistry (1,40).

Since Nth is known to excise oxidised pyrimidines and cleave apurinic/apyrimidinic (AP) sites, the fact that guanine residue damages are excised by the Nth was unexpected. Since the quantum yield of Nth excised damage at guanine sites (Table 1) is greater than the quantum yield of alkali-labile sites ([Phi] = 9.6 × 10-5) (15), it is unlikely that the only damage detected by the Nth is AP sites. Even so, AP sites, if formed, are only detected at guanine sites. Excision of damage at guanine by Nth has been previously reported for UV and also [gamma]-irradiated DNA samples (41). 2,2-Diaminooxazolone is a product of the guanosine radical cation in DNA (17) and was considered as a possible substrate for excision by Nth (Nth has a broad substrate specificity and excises ring saturated, ring opened and ring fragmented pyrimidines, which are non-planar in structure; 19,20). Even though 2,2-diaminooxazolone is formed by exposure of DNA to 193 nm light, it is not excised by Nth.

Excision of damage by Nth at guanine yields two fragments with slightly different electrophoretic mobilities in the sequencing gel. This is illustrated using the example shown in Figure 5, for the sequence AGGAAAA (labels a and b). A doublet of bands is observed for the prompt ssb in lane i (label a) and a faster running doublet for the Fpg-sensitive damage in lane ii (label b), a triplet is observed in lane iii for the Nth-sensitive damage (labels a and b). Unlike Fpg, which cleaves the AP site by a [beta],[delta]-elimination resulting in total removal of the damaged nucleoside (20), Nth cleaves the AP site by a [beta]-elimination pathway to yield a 3[prime]-[alpha],[beta]-unsaturated aldehyde terminus (19,20,42); this 3[prime]-end group is unstable under standard polyacrylamide gel electrophoresis conditions (43). Loss of some of the heat-labile 3[prime]-end groups yields a DNA fragment, which migrates through the gel to the same distance as the respective Maxam-Gilbert sequences or Fpg-treated samples. Samples with an intact 3[prime]-[alpha],[beta]-unsaturated aldehyde terminus migrate to a comparable distance to the fragments generated by prompt ssb.

The sequence dependence of the yield of 193 nm light-induced damage excised at guanine by Nth follows a similar trend as that for the prompt ssb, Fpg damage and hot alkali-labile damage. It is therefore suggested that the damage precursor is common and most likely a guanine radical cation. However, it is apparent (from Table 1) that not all the guanine radical cations (<10%) can be accounted for as frank ssb or damage excised by Fpg (18) and Nth. Even though these excision enzymes are not 100% efficient at excision (44,45), the yield of guanine damage excised from the 193 nm irradiated DNA is low. Consequently, it is suggested that the deprotonated radical cation of guanine is reconstituted as guanine (46,47) and/or further guanine damages are formed and undetected by treatment with hot alkali or by the excision enzymes used in this study. As shown in Figure 4, one adenine lesion formed by exposure of the DNA to the 193 nm light was sensitive to hot alkali but not Fpg or T4 endo V. It is suggested that 4,6-diamino-5-formamidopyrimidine (which is excised by Fpg or T4 endo V; 19) is not a major product arising from the adenine radical cation within DNA at pH 7 under aerobic conditions. This alkali-labile adenine lesion, present in the randomly sequenced MIP-1[alpha] fragment, is identified in a sequence of four adenine residues flanked by less easily oxidised cytosine and thymine residues. It is suggested that either (i) electron holes, formed within this adenine-rich region, do not migrate to a guanine residue on the paired strand or to a much more remote intrastrand guanine or (ii) one of these adenines, by virtue of its neighbouring base sequence, has an ionisation potential which is lower than that for a nearby guanine.

Other types of DNA lesions may be formed by reductive or photo pathways upon exposure to 193 nm light. Irradiation of DNA with 193 nm light results not only in the formation of oxidised DNA but also an electron. Under oxic conditions the electron is rapidly scavenged by oxygen (48). For samples irradiated under anoxic conditions, the yield of DNA electron adducts (1,49) from the reaction of the solvated electron with DNA is estimated to be <1% for DNA [the lifetime of the electron under our conditions is ~5 µs, [DNA] ~ 0.5 µmol/dm3, k(e-(aq) + DNA) = 1.4 × 108 dm3/mol/s; 50]. Consistent with this, there is not an oxygen effect on the yield of Nth-sensitive sites (Nth is known to excise both dihydrothymine and dihydrouracil; 19). Furthermore, dihydropyrimidines are not detected by GC/MS in a DNA sample irradiated under anoxic conditions by 193 nm light (T.Melvin and M.Dizdaroglu, unpublished results).

Since high quantum yields of photohydrates are detected in DNA irradiated with UV-B or UV-C light, pyrimidine photohydrates might have been expected from 193 nm irradiation of DNA (11,21,22). However, these lesions if formed and excised by Nth, are present in far lower quantities than lesions detected at guanine by Nth. The yield of cyclobutylpyrimidine dimers induced by irradiation of plasmid DNA with 193 nm light and excised by the Micrococcus UV endonuclease was previously determined to be 1.65 × 10-3 in air saturated solutions (15,34). This yield of photodimers is slightly greater than reported here using T4 endo V (air 5.5 × 10-4, argon 8.8 × 10-4). Further, T4 endo V does not excise any damage other than pyrimidine dimers. It had been suggested by Gut et al. (34) that excitation of DNA bases to the singlet manifold yields pyrimidine dimers and excitation to higher excited states results in photoionisation and the subsequent formation of alkali-labile sites. We suggest that excitation of DNA with 193 nm light results in low yields of photoionisation (Table 1) and the majority of the photon energy must be lost via processes which bypass the excited states responsible for the formation of photodimers and hydrates. If higher excited states are involved, the lifetimes would be expected to be exceedingly short (~1 ps) (51 and references therein), allowing only reaction with neighbouring bases and solvent, and again this is not observed.

In summary, irradiation of DNA with 193 nm light results in damage, which is localised predominantly at guanine; the yield of photo-damage such as pyrimidine dimers is relatively minor at this wavelength. Photoionisation of DNA yields radical cations of all the nucleic acid bases; the subsequent migration to the most easily oxidised base, guanine, occurs and results in `targeted' damage at this site. These results indicate that damage at guanine and the sequence-dependent yields are a signature for the oxidative pathways of direct ionisation, which is a major component of the damaging effect of exposure of cellular systems to ionising radiation (1).

ACKNOWLEDGEMENTS

The authors would like to acknowledge the Rutherford Appleton Laboratory (Lasers for Science Facility) for help with the excimer laser and thank Prof. A.A.van Zeeland and A.van Hoffen (University of Leiden, The Netherlands) for the gift of pure T4 endo V enzyme. Funded by the Commission of the European Community, Contract no. F14-CT95-0011c.

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*To whom correspondence should be addressed at present address: Centre de Biophysique Moleculaire CNRS, Rue Charles Sadron, 45071 Orléans Cedex 2, France. Tel: +33 238 25 55 40; Fax: +33 238 63 15 17; Email: melvin@cnrs-orleans.fr

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