Mutation Research/DNA Repair
Oxidative DNA base damage induced by singlet oxygen and photosensitization: recognition by repair endonucleases and mutagenicity
Introduction
Oxidative guanine modifications are the prevailing type of damage generated by apparently all types of reactive oxygen species (ROS), since this base has the lowest one-electron oxidation potential [1]. A highly selective generation of guanine modifications has been well-established for photosensitizers which modify DNA either via singlet oxygen (type-II photoreaction) or one-electron oxidation (type-I photoreaction) [2], [3], [4], [5]. As expected, the selectivity is less pronounced for the most reactive hydroxyl radicals, which generate relatively high amounts of single-strand breaks (SSB), sites of base loss (AP sites) and modifications of other bases as well [6], [7], [8], [9].
Among the guanine modifications, 7,8-dihydro-8-oxoguanine (8-oxoGua) is best-known, and its generation in cellular and cell-free DNA was demonstrated for virtually all types of ROS [10], [11], [12], [13]. The lesion has a distinct mutagenic potential, giving rise to GC→TA transversion [14], [15], [16]. Other guanine modifications that have been identified after reaction of DNA or isolated nucleotides with many oxidants include the imidazol-ring opened formamidopyrimidine FapyGua, the 2,2,4-triaminooxazolone Z and its precursor, the imidazolone Iz, and 4-hydroxy-8-oxo-guanine [8], [17] (Fig. 1). Until now, there is only little knowledge about the relative yields of the various guanine modifications generated by ROS. Analytical problems arise not only from the limited sensitivity of the detection methods for some of the modifications, but also from an artifactual oxidation during the sample preparation [18], [19] and from the fact that the secondary oxidation of 8-oxoGua (and possibly other oxidative guanine modifications) already becomes prominent at those levels of damage that are required for the quantification of most other types of lesions [20].
To prevent the deleterious consequences of oxidative DNA damage for the integrity of the genome, both prokaryotic and eukaryotic cells have developed specific repair mechanisms [21], [22], [23], [24]. In bacteria, the removal of oxidative guanine and adenine modifications is initiated by Fpg protein, an endonuclease which has been shown to recognize 8-oxoGua and FapyGua, besides AP sites and several other lesions (Table 1) [25], [26], [27]. Complementary, oxidative pyrimidine modifications are substrates of endonuclease III and endonuclease VIII [28], [29], [30], [31], [32]. In eukaryotic cells, 8-oxoGua and FapyGua are recognized by the Ogg1 protein, which share no sequence homology with the bacterial Fpg protein [33], [34]. Functional and also structural homologues of endonuclease III in eukaryotes are Ntg1 and Ntg2 proteins in yeast [35], [36] and Nth1 protein in mammalian cells [37], [38], [39]. Ntg1 localizes primarily to the mitochondria [35], [40]. The spectrum of substrate modifications of these eukaryotic enzymes seems to overlap largely with that of endonuclease III (Table 1). A major difference is that the purine-derived formamidopyrimidines were found to be the good substrates of Ntg1 and Ntg2 proteins, but not of endonuclease III [35], [41], [42], [43].
In the study described here, we compared the recognition by the above-mentioned enzymes of the base modifications induced by three oxidants, viz. [4-(tert-butyldioxycarbonyl)benzyl]triethylammonium chloride (BCBT), a photochemical source of tert-butoxyl radicals [44], disodium salt of 1,4-etheno-2,3-benzodioxin-1,4-dipropanoic acid (NDPO2), which generates singlet oxygen by thermal decomposition [45], and photoexcited riboflavin, which reacts with DNA by one-electron oxidation [46]. The results not only revealed new distinct differences between the recognition spectra of the prokaryotic and eukaryotic enzymes, but also indicate that the spectrum of guanine modifications induced by the oxidants differs considerable.
Section snippets
Materials
DNA from bacteriophage PM2 (PM2 DNA) was prepared according to the method of Salditt et al. [47]. More than 97% was in the supercoiled form, as determined by the method described below. Formamidopyrimidine-DNA glycosylase (Fpg protein) from Escherichia coli was purified as described previously [25]. Endonuclease III was purified from the overproducing E. coli strain BH410 (fpg-1) [48] harboring the pNTH10 plasmid. Ogg1 protein from Saccharomyces cerevisiae was purified from E. coli BH410 (fpg-1
Recognition of AP sites
Sites of base loss (AP sites) are common substrates of all repair endonucleases and therefore are suitable reference modifications for comparisons. We used supercoiled bacteriophage PM2 partially DNA depurinated by exposure to pH 4.5 for 6 min at 70°C (AP-DNA) as a model substrate to quantify the recognition of AP sites by the various bacterial and eucaryotic repair endonucleases used in this study (Table 1) by means of a relaxation assay.
The results (Fig. 2, upper panel) indicate that
Discussion
The results presented here indicate that at least three types of oxidative guanine modifications are generated in DNA under conditions in which secondary damage of performed modifications is unlikely to play a role (induction of less than one modification per 104 bp). The modifications can be distinguished by their recognition by repair endonucleases. The first modification, 8-oxoGua, is recognized by Fpg protein and its eukaryotic functional analogue Ogg1, but not by Ntg1 and Ntg2 proteins, in
Acknowledgements
This study was supported by the Deutsche Forschungsgemeinschaft (SFB 519) and by the Commissariat à l’Energie Atomique (CEA).
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