A study of endonuclease III-sensitive sites in irradiated DNA: detection of
-particle-induced oxidative damage
Kevin M. Prise1,
Clare H.L. Pullar and
Barry D. Michael
Gray Laboratory Cancer Research Trust, PO Box 100, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR, UK
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Abstract
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An important difference between chemical agents that induce oxidative damage in DNA and ionizing radiation is that radiation-induced damage is clustered locally on the DNA. Both modelling and experimental studies have predicted the importance of clustering of lesions induced by ionizing radiation and its dependence on radiation quality. With increasing linear energy transfer, it is predicted that complex lesions will be formed within 120 bp regions of the DNA. As well as strand breaks, these sites may contain multiple damaged bases. We have compared the yields of single strand breaks (ssb) and double strand breaks (dsb) along with those produced by treatment of irradiated DNA with the enzyme endonuclease III, which recognizes a number of oxidized pyrimidines in DNA and converts them to strand breaks. Plasmid DNA was irradiated under two different scavenging conditions to test the involvement of OH· radicals with either 60Co
-rays or
-particles from a 238Pu source. Under low scavenging conditions (10 mM Tris)
-irradiation induced 7.1x107 ssb Gy/bp, which increased 3.7-fold to 2.6x106 ssb Gy/bp with endo III treatment. In contrast the yields of dsb increased by 4.2-fold from 1.5x108 to 6.3x108 dsb Gy/bp. This equates to an additional 2.5% of the endo III-sensitive sites being converted to dsb on enzyme treatment. For
-particles this increased to 9%. Given that endo III sensitive sites may only constitute ~40% of the base lesions induced in DNA, this suggests that up to 6% of the ssb measured in X- and 22% in
-particle-irradiated DNA could have damaged bases associated with them contributing to lesion complexity.
Abbreviations: BSA, bovine serum albumin; dsb, double strand breaks; endo III, endonuclease III; FPG, formamidopyrimidineDNA N-glycosylase; GCMS, gas chromatographymass spectrometry; LET, linear energy transfer; RBE, relative biological effectiveness; ssb, single strand breaks.
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Introduction
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DNA can be damaged by a range of oxidative agents and, in the cellular situation, it is continually being oxidatively damaged by normal metabolic processes (1). For ionizing radiation, damage is predominantly produced via OH· radicals (2). A key difference between ionizing radiation-induced damage and that produced by other chemical oxidizing agents is that a range of damage products are produced and these are locally clustered on the DNA (3). Given this background, understanding the effectiveness of different types of ionizing radiation, with varying linear energy transfer (LET) values, in biological systems has been an area of intense study. Understanding the carcinogenic risk associated with environmental exposure to
-particles from radon gas requires knowledge of the lesions induced in DNA if studies of cellular mechanisms of DNA repair and carcinogenesis are to be undertaken. At the level of the cellular DNA, biophysical modelling studies have predicted that not only the yields, but the complexity of the damage produced are important (4,5). With increasing LET, it is predicted that increasing clustering of lesions occurs locally on the DNA within 120 bp. These will consist of the different types of lesions including base and sugar damage, double strand breaks (dsb), single strand breaks (ssb), DNADNA and DNAprotein cross-links. Although DNA dsb have been predicted to be important lesions in terms of biological effectiveness, they may simply be markers for the production of clustered damage. To date, little evidence has been obtained for the association of base damage with clustered lesions and the role of radiation quality in the degree of clustering. Base damage induced by oxidative processes has been shown to play a role in mutagenesis and carcinogenesis (see 6, for a review). For ionizing radiation, it has been predicted that damaged bases are present in greater yields than strand breaks. Values for the ratio of base to sugar damage varies from 2.7 (5), if one considers the likely radiation chemistry of indirect damage that may occur, to values of ~20 based on measurements of oxidized bases in cellular systems (7,8). Although recent studies have shown that some measurements may be overestimated due to oxidation of bases during the derivatization step required for gas chromatographymass spectrometry (GCMS) studies (9,10). A few enzymes are available that recognize oxidized bases in DNA and convert them to strand breaks, which can then be detected by in vitro assays. Of these, endonuclease III (endo III) and formamidopyrimidineDNA N-glycosylase (FPG) have been the most extensively characterized. Treatment of DNA with these enzymes allows estimates of the yields of base damage to be determined both in model DNA (11,12) and cellular systems (1316). In this short study, we have determined the additional strand breakage exposed in DNA after irradiation with
-rays or high LET
-particles by treatment with endo III. Endo III is a class I AP endonuclease with an associated N-glycosylase activity specific for a number of oxidized pyrimidine residues (11,12). We have measured the yields of ssb and dsb as well as the additional yields of these lesions produced by treatment with endo III under two differing scavenging conditions, with the view of gaining additional information regarding the association of base damage with complex lesions.
Plasmid pMSG-CAT DNA (8405 bp) was purified from Escherichia coli strain HB101 using a commercially available kit (Qiagen, Dorking, UK). The DNA was stored in TE buffer (10 mmol/dm3 Tris, 1 mmol/dm3 EDTA, pH 7.5) at a concentration of 0.5 µg/µl. For experiments, the DNA was diluted to 0.1 µg/µl and Tris added to give a final concentration of 10 or 200 mol/dm3. For
-irradiation, DNA equilibrated in air, was irradiated in glass reaction vials (Pierce Scientific, UK) with 60Co
-rays at a dose-rate of ~20 Gy/min. For irradiation with
-particles, 2.65 µl aliquots of plasmid solution were placed under a glass coverslip on a mylar based dish (3 µm thick) and exposed to 3.5 MeV
-particles (110 keV/µm) through the mylar foil from a 238Pu source at a dose-rate of 54 Gy/min.
Purified endo III was kindly provided by Dr A.R.Collins (University of Aberdeen, UK). In some experiments commercially available endo III was used (Trevigen, Gaithersburg, USA) and gave identical results. After irradiation, samples were incubated in the presence of 1 unit of endo III (1 unit of enzyme is defined as the cleavage at an AP site of an oligonucleotide at a rate of 1 pmol/h) at 37°C for 45 min in the presence of 40 mmol/dm3 HEPES, 0.1 mol/dm3 KCl, 0.5 mmol/dm3 EDTA, 0.2 mg/ml bovine serum albumin (BSA), pH 8.0. Control samples were also incubated in the presence of incubation buffer only. Reactions were stopped by the addition of gel-loading buffer [0.25% (w/v) bromophenol blue, 20% (w/v) Ficoll 400, 0.1 mol/dm3 EDTA] and held at 4°C until electrophoresis. Samples of DNA were separated on 0.8% (w/v) agarose gels in TBE buffer (0.089 mol/dm3 Tris, 0.089 mol/dm3 boric acid, 2 mmol/dm3 EDTA, pH 8.0) at 0.9 V/cm for 16 h. After electrophoresis, gels were stained in TBE containing 0.5 µg/ml ethidium bromide, destained and viewed on a 302 nm transilluminator. Images of the gels were captured using a custom-built image capture system consisting of a 8-bit CCD camera (COHU), image capture board (Data Translations DT55-50 Hz, 8-bit 786x512 pixels image capture) and an in-house developed analysis package (based on Visilog software, Datacell, UK). Three forms of plasmid DNA were observed: supercoiled (form I), relaxed (form II) and linear (form III). The value obtained for the supercoiled form was corrected, by a factor of 1.2, for the reduced binding of ethidium bromide into this form. This correction was calculated by comparing the fluorescence from known amounts of the three plasmid forms.
Plasmid pMSG-CAT was exposed to
-rays or
-particles and then incubated with and without endo III, and the yields of ssb and dsb measured using gel electrophoresis. Preliminary experiments tested the effect of increasing amount of enzyme and incubation time on the additional yields of breaks produced by endo III incubation. Different amounts of enzyme (020 U) were added after irradiation with 100 Gy
-rays in the presence of 200 mM Tris. In general, at 1 U and above, saturation occurred. Using 1 U enzyme as standard, the optimal incubation time was ascertained to be 45 min and this condition was used for all subsequent enzyme incubations. For all the conditions studied, no significant effect of endo III incubation on the background levels of damage was observed (results not shown). Doseresponse curves were then obtained for DNA irradiated with 60Co
-rays or
-particles exposed under two different concentrations of Tris corresponding to scavenging capacities of 1.5x107/s (10 mM) and 3x108/s (200 mM). These equate to OH· diffusion distances of 28 and 6 nm, respectively (17). The upper panels of Figure 1
show the loss of the supercoiled form of the plasmid versus dose, which is a measure of the production of ssb in the molecule. Treatment with endo III leads to an increase in the yields of ssb being detected over the whole dose range. The lower panel depicts the production of the linear form of the plasmid, which is a measure of dsb formed in the molecule. Again, over the whole dose range, incubation with endo III led to increased yields of dsb. From these data, linear regression fits were performed to measure the yields of breaks induced (Table I
) under these conditions (18).
By increasing the concentration of scavenger present it is possible to reduce the role of OH·-mediated damage on the DNA. Given that many studies have been performed using a range of scavenger concentrations, only a limited range was considered here. For
-irradiated DNA this leads, in the absence of endo III treatment, to an ~2-fold decrease in the yields of both ssb (from 7.1x107 to 4.2x107 ssb Gy/bp) and dsb (from 1.5x108 to 7.1x109 dsb Gy/bp) as has been reported by many other studies (1921). A similar ~2-fold reduction in the yields of breaks is observed for
-particles with a reduction in the yield of ssb from 2.0x107 to 7.6x108 dsb Gy/bp and dsb from 2.5x108 to 1.5x108 dsb Gy/bp as found by other groups (22,23). This decrease is in line with the decreased yield of OH· with increasing LET (24). A similar difference is observed in the endo III treated DNA with increasing scavenger concentration, which suggests that these damaged bases are produced by the action of OH·. These data are in line with studies which have shown that the major base products that are recognized by endo III are detectable in irradiated DNA. The increase in the yields of ssb after endo III treatment by a factor of 3 are similar to those found by Milligan et al. (25) in a more extensive study over a large scavenger range. Importantly, however, the relative increase in ssb versus dsb for endo III treatment are different with a greater proportion of additional dsb being induced (a factor of 4.2 ± 0.5 for dsb versus 3.7 ± 0.4 for ssb at 10 mM and 5.2 ± 0.4 for dsb versus 4.5 ± 0.2 for ssb at 200 mM) (see Table I
). The relative changes we have observed between these two scavenger concentrations are less than that found by other researchers but may reflect differences in the concentration of DNA used as it will scavenge radicals also.
The additional dsb we detected in this system with treatment of endo III can come from two sources when only simple lesions consisting of one damaged base on each strand are considered. These are cleavage by endo III of a damaged base near to an existing ssb on the opposite strand or, two damaged bases on opposite strands close enough to form a dsb when recognized by endo III. The combined proportions of these can be determined by expressing the yield of additional dsb produced by endo III treatment as a percentage of the additional ssb produced by endo III treatment. For
-irradiated DNA this accounts for 2.5% of the additional endo III-sensitive damaged bases at 10 mM [i.e. (6.3x108 1.5x108) / (2.6x106 7.1x107)x100] and 2% at 200 mM (see Table I
). The fact that the yields are linear with dose rules out these additional breaks being caused by the increased probability of endo III-induced ssb being close enough together on opposite strands of the DNA to produce a dsb indirectly. Breaks of this nature would be expected to increase quadratically with dose.
The biggest differences we observed were when comparing two different forms of radiation. For high LET
-particles we observe marked differences in the induction of both ssb and dsb in comparison with
-rays. The relative biological effectiveness (RBE) is 1.7 ± 0.2 and 2.1 ± 0.2 for dsb at 10 and 200 mM Tris, respectively, but 0.28 ± 0.04 and 0.18 ± 0.01 for ssb in the absence of endo III. This is normally taken to mean that the yield of solo ssb is decreasing and a greater proportion of the remaining ssb are associated with clustered damage and are included in the yields of dsb. This agrees with the decrease in the ratio of ssb to dsb from ~50:1 for
-rays to ~6:1 for
-particles and is in line with studies showing that the yield of OH· decreases with increasing LET (22,23) and the complexity of lesions produced increases. For
-particles, the treatment of irradiated DNA with endo III leads to both additional ssb and dsb at both scavenger concentrations. However, the enhancement of yields are lower than that observed with
-rays, with enhancement values of 1.8 ± 0.2 for ssb and 1.6 ± 0.1 for dsb at 10 mM, and 2.5 ± 0.2 for ssb and 1.3 ± 0.1 for dsb at 200 mM. Overall, however, the percentage of additional endo III-sensitive breaks associated with dsb is higher than with
-rays, giving values of 9% for 10 mM Tris and 4% for 200 mM Tris.
Underlying the detection of these complex lesions will be two factors, firstly the ability of endo III to recognize and excise damaged bases close together on opposite strands of the DNA and secondly, how close two ssb on opposite strands need to be for a dsb to be detected. Recent studies have shown that more complex lesions are less likely to be digested with endo III (26). The glycosylase activity of the enzyme, but not its lyase activity, is inhibited by a closely positioned break in the opposite strand. The authors used substrates containing thymine glycol residues spaced 1, 3, 5 and 7 bases apart on opposite strands. They found that substrates where the lesions were only 13 bp apart yielded only ssb whereas the other substrates yielded dsb. This suggests that very complex lesions where individual damaged bases are very close together in our system may not be detected and may explain the decrease in these with increasing LET and the increase in the ratio of single to double endo III-sensitive sites. Recently, evidence has also been obtained for the production of tandem base damage, i.e. two neighbouring damaged bases on the same strand of DNA (27). The probable consequences of such damage in terms of radiation effects or the ability of enzymes such as endo III to recognize these sites has not been determined. These lesions may be produced from a single OH· and not as a result of two independent events.
To our knowledge, very little has been reported regarding the induction of base damage by high LET radiation (28). Only one study (29) has measured specific base modifications. They found that in cells irradiated with carbon-ions, the yields of 5,6-dihydrothymine decreased by a factor of 5 in comparison with X-rays. The overall decrease in endo III-sensitive sites in
-particle irradiated DNA in comparison with
-rays observed here is ~12-fold. One other study (30), in SV40 DNA irradiated in dilute solution, showed that the ratio of alkali-sensitive sites to total strand breaks increased in proportion with LET. They found that it increased from 1.5, with low LET
-rays, to 1.8 with neon ions having an LET of 115 keV/µm. Alkali-sensitive lesions represent a broad class of lesions however, which involve a few base lesions and oxidized sugar residues and thus may be limited in their applicability to mechanistic studies.
Estimates for the total yields of base damage in cellular systems are controversial principally because of concerns over the basal levels of base damage being measured using various techniques (reviewed in 31), particularly GCMS (32). The higher values previously measured with GCMS have been shown to be overestimated because of oxidation of bases during the derivatization process (9,10). Overall, however, the relative levels of base damage types may not be different. From our studies we can make some preliminary estimates of the probable level of complex lesions that contain any base damage. In isolated calf thymus, DNA irradiated in air by
-rays at 0.5 mg/ml in 30 mM phosphate buffer, thymine glycol and cytosine glycol constituted ~40% of the damaged base products measured. (33). Other studies have shown that FPG-sensitive sites constitute a greater proportion of additional breaks (~1.6 more) than endo III-sensitive ones (16,34). Assuming that the distributions of different base lesions are the same, this suggests that with low LET
-rays from the 2.5% of complex endo III sites that we have measured, a minimum of ~6% of the complex lesions would have contained damaged bases if we could have detected them all. From the 9% of endo III complex lesions we measured for
-particles this would translate to ~22% in total. These values may represent a lower limit for the involvement of damaged bases with complex lesions, as some complex lesions may not be detected in this study (26). Endo III also recognizes sites of base loss (AP sites). Although these may be induced at frequencies similar to that of strand breakage, around 5070% of these are associated with the sites of strand breaks (34,35). Recently, in a similar study by Milligan et al. (25), treatment of irradiated DNA under similar scavenging conditions to those used here, with both endo III and FPG gave an additive effect compared with the exposure of the DNA to each enzyme individually. As both enzymes recognize sites of base loss, this suggests that under the conditions used here, they may not be significant. The authors suggested that these sites might be labile under conditions of agarose gel electrophoresis (36). Overall, the effects of densely ionizing radiation on the yields of single base damage measured (i.e. additional ssb) decreases with both LET and scavenger concentration, which is consistent with the reduced role of OH·-mediated damage and the increased association of the remaining endo III-sensitive sites with complex lesions.
In conclusion, we have obtained evidence for the increased association of oxidative base damage with lesions formed by the action of
-particles on plasmid DNA. These may ultimately play a role in the increased carcinogenic risk associated with these types of radiation.
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Acknowledgments
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This work was supported by the Cancer Research Campaign and the Radiation Protection Research Action Programme of the European Community. The authors are grateful to Dr A.R.Collins for providing the endo III.
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Notes
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1 To whom correspondence should be addressed Email: prise{at}graylab.ac.uk 
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Received July 22, 1998;
revised January 8, 1999;
accepted January 11, 1999.