Arabidopsis thaliana Ogg1 Protein Excises 8-Hydroxyguanine and 2,6-Diamino-4-hydroxy-5-formamidopyrimidine from Oxidatively Damaged DNA Containing Multiple Lesions

Abstract

A functional homologue of eukaryotic Ogg1 proteins in the model plant Arabidopsis thaliana has recently been cloned, isolated and characterized [Garcia-Ortiz, M. V., Ariza, R. R., and Roldan-Arjona, T. (2001) Plant Mol. Biol. 47, 795-804]. This enzyme (AtOgg1) exhibits a high degree of sequence similarity in several highly conserved regions with Saccharomyces cerevisiae, Drosophila melanogaster and human Ogg1 proteins. We investigated the substrate specificity and kinetics of AtOgg1 for excision of modified bases from oxidatively damaged DNA that contained multiple pyrimidine- and purine derived lesions. Two different DNA substrates prepared by exposure to ionizing radiation in aqueous solution under N2O or air were used for this purpose. Gas chromatography/isotope-dilution mass spectrometry was applied to identify and quantify modified bases in DNA samples. Of the seventeen modified bases identified in DNA samples, only 8-hydroxyguanine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine were significantly excised from both DNA substrates. This is in agreement with the substrate specificities of other eukaryotic Ogg1 proteins that had previously been studied under identical conditions. Excision depended on incubation time, enzyme concentration and substrate concentration, and followed Michaelis-Menten kinetics. A significant dependence of excision on the nature of DNA substrate was observed in accord with previous studies on other DNA glycosylases. A comparison of excision kinetics pointed to significant differences between AtOgg1 and other Ogg1 proteins. We also investigated the effect of base-pairing on the excision using double-stranded oligodeoxynucleotides that contained 8-OH-Gua paired with each of the four DNA bases. The activity of AtOgg1 was most effective on the 8-OH-Gua:C pair with some or very low activity on other pairs in agreement with the activity of other Ogg1 proteins. The results unequivocally show that AtOgg1 possesses common substrates with other eukaryotic Ogg1 proteins albeit significant differences between their excision kinetics. Oxygen-derived species such as free radicals and other oxidizing agents generate a multiplicity of lesions in DNA comprising modified bases and sugars, DNA-protein cross-links, strand breaks and base-free sites (reviewed in ref (1;2)). A variety of repair pathways exist in cells to combat DNA damage and to maintain genomic integrity (reviewed in ref (3)). The repair of modified bases in DNA of both eukaryotes and prokaryotes primarily occurs via the base-excision repair (BER)1 pathway (reviewed in ref (4)), which is conserved throughout all species. DNA glycosylases are involved in the first step of BER and remove modified bases from DNA by catalyzing the hydrolysis of the glycosidic bond between the modified base and the sugar moiety. In Escherichia coli, formamidopyrimidine DNA glycosylase (Fpg) is a DNA glycosylase/lyase that excises 8-hydroxyguanine (8-OH-Gua), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4,6-diamino-5-formamidopyrimidine (FapyAde) from DNA and processes the resulting abasic site by cleaving both 3´- and 5´-phosphodiester bonds by successive β- and δ-eliminations (5-7). Fpg shows a clear preference for Cyt as the base opposite 8-OH-Gua, with 8-OH-Gua:Ade being a particularly poor substrate (8). This specificity avoids repair of 8-OH-Gua:Ade mispairs to T:A, which would cause a GC→TA transversion. The repair of 8-OH-Gua:Ade mispairs is initiated instead by MutY, a DNA glycosylase that catalyze excision of misincorporated Ade (9). Animals and yeast cells possess 8-OH-Gua DNA glycosylases that do not share significant sequence identity with bacterial Fpg proteins. The first of these Fpg analogues was identified in Saccharomyces cerevisiae and designated yOgg1 (10;11). This enzyme possesses a similar substrate specificity to Fpg and removes 8-OH-Gua and FapyGua, but not FapyAde in contrast to Fpg (12), and displays a preference for 8-OH-Gua opposite Cyt, but not Ade (10;11;13). Genes encoding proteins, which share significant sequence identity with yOgg1, have been subsequently cloned and characterized in humans and other mammals (14-20). Genome analyses revealed proteins similar to Ogg1 in Archaea but not in any bacterial species (21). Arabidopsis thaliana has become a very attractive model system for study of conserved DNA repair pathways due to the practical advantages of small size and rapid life cycle in conjunction with the recent development of powerful tools to study its genome. In addition, there are important differences between the life strategies of plants and most eukaryotes. For example, plants do not have a reserve germ line, and their gametes differentiate late in development from somatic cells. This and other differences may shed light on crucial aspects of genome-maintenance functions in eukaryotes. We recently isolated and characterized an Ogg1 orthologue in the model plant Arabidopsis thaliana (AtOgg1) (22). Our finding was of particular interest, since an 8-OH-Gua DNA glycosylase (AtMMH) encoded by a gene named AtMMH, which is an orthologue of E. coli’s gene Fpg, had previously been isolated and characterized in this model plant (23). The discovery of AtOgg1 established plants as the only organisms, where the presence of both Fpg and Ogg1 homologues exist. On the other hand, Arabidopsis is not the only example of an organism with two different enzymes for the repair of 8-OH-Gua. In Drosophila melanogaster, both the ribosomal protein S3 and an Ogg1 protein (dOgg1), which is a true orthologue of other Ogg1 proteins, possess DNA glycosylase/β-lyase activity capable of releasing 8-OH-Gua and FapyGua from damaged DNA with multiple lesions (24;25). In the case of Arabidopsis, the relative roles of AtOgg1 and AtMMH might be connected to their likely different phylogenetic origin, since the latter may be the result of a gene transfer from an ancestral chloroplast genome to the nucleus (23). Although there are putative nuclear targeting signals in the predicted amino acid sequences of AtOgg1 and AtMMH, the subcellular localization of both enzymes remains to be determined. The possibility exists that AtOgg1 removes modified bases for oxidatively damaged DNA in the nucleus, while AtMMH continues performing an essential DNA repair activity in the chloroplast genome. In the present work, we report the substrate specificity and excision kinetics of AtOgg1 using oxidatively damaged DNA containing a multiplicity of lesions. Excision by AtOgg1of modified DNA bases was studied using the technique of gas chromatography/isotope-dilution mass spectrometry (GC/IDMS). Furthermore, an oligodeoxynucleotide containing a single 8-OH-Gua residue at a defined position, which was paired with each of the four DNA bases, were used to investigate the paired-base effect on the repair activity of this protein.