Abstract
Reactive oxygen species (ROS) generated during energy production processes are a major cause of oxidative DNA damage. A DNA glycosylase encoded by the Ogg1 gene removes oxidized guanine bases and is widely conserved. However, the biological role of the gene in individual organisms has not yet been characterized in Drosophila, which is a suitable model to study the influence of oxidative damage on senescence. Here, we performed a genetic analysis to confirm that Ogg1 plays an essential role in the removal of 8-oxo-guanines from nuclei. We first confirmed by quantitative real-time PCR that Ogg1 mRNA expression was reduced by 30-55% in Ogg1 mutants and in flies expressing inducible Ogg1 dsRNA compared to control flies. We then showed that additional accumulation of 8-oxo-guanines occurred in the nuclei of epithelial midgut cells after paraquat feeding in flies with downregulated Ogg1 expression. We confirmed that a transposon possessing the UAS sequence was integrated in the 5'-UTR of the Ogg1 alleles and that it is oriented in the same transcriptional direction as the gene. Using the Gal4/UAS system, which enables us to induce ectopic expression in Drosophila, we induced overexpression of Ogg1 by 40-fold. We observed a lower amount of 8-oxo-guanine in the midgut epithelial cells of adults overexpressing Ogg1. These genetic data strongly suggest that the Drosophila Ogg1 ortholog CG1795 plays an essential role in the suppression of 8-oxo-guanines, consistent with its role in other organisms. Although adult flies with reduced Ogg1 expression failed to show elevated sensitivity to paraquat, those with Ogg1 overexpression showed resistance to oxidative stress by paraquat feeding and had a significantly longer lifespan in normal feeding conditions. These observations are consistent with the hypothesis that oxidative DNA damage by ROS accumulation is a major contributor to senescence.
Highlights
Reactive oxygen species (ROS) induce damage in genomic as well as mitochondrial DNA (Lindahl, 1993; Wang et al, 1998; Cooke et al, 2003)
Reduced expression of Drosophila Ogg1 in transposon-induced mutants and in flies depleted of Ogg1 mRNA by induced dsRNA expression To examine whether the D. melanogaster gene CG1795, encoding an Ogg1 ortholog, is required for the removal of oxidized guanine bases in the DNA repair process, we examined the accumulation of 8-oxo-guanines in transposon-induced mutants of the Ogg1 gene
Our quantitative real-time PCR analysis revealed that Ogg1 mRNA expression was less than 60% of the level observed in the controls for both insertional mutants (Fig. 1B)
Summary
Reactive oxygen species (ROS) induce damage in genomic as well as mitochondrial DNA (Lindahl, 1993; Wang et al, 1998; Cooke et al, 2003). The production of oxidized bases can lead to DNA mutagenesis, and the accumulation of such DNA damage eventually results in the development of many diseases and senescence (Barja, 2004; Maynard et al, 2009; Kryston et al, 2011). To prevent these effects, oxidized DNA bases are initially removed by the base-excision repair mechanism (Wallace, 1998; Hazra and Mitra, 2006; Nemec et al, 2010). First discovered in Escherichia coli, the DNA glycosylase MutM excises 8-oxo-guanines generated by ROS (Michaels et al, 1992; Grollman and Moriya, 1993; Boiteux and Radicella, 1999). It was shown that the Ogg ortholog in human cells is a major enzyme to repair 8-oxoguanine DNA lesions (Monden et al, 1999; Boiteux and Radicella, 2000)
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