In vivo repair of alkylating and oxidative DNA damage in the mitochondrial and nuclear genomes of wild-type and glycosylase-deficient Caenorhabditis elegans

Abstract

Base excision repair (BER) is an evolutionarily conserved DNA repair pathway that is critical for repair of many of the most common types of DNA damage generated both by endogenous metabolic pathways and exposure to exogenous stressors such as pollutants. Caenorhabditis elegans is an increasingly important model organism for the study of DNA damage-related processes including DNA repair, genotoxicity, and apoptosis, but BER is not well understood in this organism, and has not previously been measured in vivo. We report robust BER in the nuclear genome and slightly slower damage removal from the mitochondrial genome; in both cases the removal rates are comparable to those observed in mammals. However we could detect no deficiency in BER in the nth-1 strain, which carries a deletion in the only glycosylase yet described in C. elegans that repairs oxidative DNA damage. We also failed to detect increased lethality or growth inhibition in nth-1 nematodes after exposure to oxidative or alkylating damage, suggesting the existence of at least one additional as-yet undetected glycosylase.

Figure 1

N2 and nth-1 C. elegans show robust nuclear DNA repair following damage induced by 5mM H₂O₂ or 5 mM MMS. Nuclear DNA damage was analyzed following acute exposure of L1 larvae to H₂O₂ and MMS (0h). Subsequent repair was measured 3, 6, or 24h post-exposure. n = 6–11 nematodes per toxin per time point. Error bars represent the standard error of the mean.

Figure 2

N2 and nth-1 C. elegans show efficient repair of H₂O₂ and MMS-induced mitochondrial DNA damage. Mitochondrial DNA damage was measured in N2 (wild-type) and nth-1 C. elegans using QPCR following exposure of L1 larvae to 5mM H₂O₂ or 5mM MMS (time 0h). Repair of mitochondrial DNA was analyzed 3, 6, or 24h post-exposure. n = 6–11 nematodes per toxin per time point. Error bars represent the standard error of the mean.

Figure 3

mtDNA copy number is relatively stable over 24h in N2 and nth-1 C. elegans strains exposed to 5mM H₂O₂ or 5mM MMS. Mitochondrial DNA copy number was measured in N2 (wild-type) and nth-1 C. elegans using quantitative, real-time PCR following exposure of L1 larvae to H₂O₂ and MMS. Data represents the relative copy number in samples analyzed 3, 6, or 24h post-exposure as compared to the 0h time point. n = 6–11 nematodes per toxin per time point. Error bars represent the standard error of the mean.

Figure 4

Growth inhibition by hydrogen peroxide (1.5 mM) and MMS (2.5 mM) is indistinguishable in the N2 and nth-1 strains. n = 74–334 nematodes per time point per strain per chemical; results include four separate (pooled) experiments.