Nucleotide excision repair genes are expressed at low levels and are not detectably inducible in Caenorhabditis elegans somatic tissues, but their function is required for normal adult life after UVC exposure

Abstract

We performed experiments to characterize the inducibility of nucleotide excision repair (NER) in Caenorhabditis elegans, and to examine global gene expression in NER-deficient and -proficient strains as well as germline vs. somatic tissues, with and without genotoxic stress. We also carried out experiments to elucidate the importance of NER in the adult life of C. elegans under genotoxin-stressed and control conditions. Adult lifespan was not detectably different between wild-type and NER-deficient xpa-1 nematodes under control conditions. However, exposure to 6J/m(2)/day of ultraviolet C radiation (UVC) decreased lifespan in xpa-1 nematodes more than a dose of 100 J/m(2)/day in wild-type. Similar differential sensitivities were observed for adult size and feeding. Remarkably, global gene expression was nearly identical in young adult wild-type and xpa-1 nematodes, both in control conditions and 3h after exposure to 50 J/m(2) UVC. Neither NER genes nor repair activity were detectably inducible in young adults that lacked germ cells and developing embryos (glp-1 strain). However, expression levels of dozens of NER and other DNA damage response genes were much (5-30-fold) lower in adults lacking germ cells and developing embryos, suggesting that somatic and post-mitotic cells have a much lower DNA repair ability. Finally, we describe a refinement of our DNA damage assay that allows damage measurement in single nematodes.

Figure 1. NER is required for normal lifespan of UVC-exposed adult C. elegans

Wild-type (red) and xpa-1 (blue) adult lifespans after chronic (daily) exposures to 0, 6, 12, 25, 50, or 100 J/m² UVC irradiation. The average of 2 replicate experiments is presented.

Figure 2. Images of phenotypes observed during lifespan studies

Wild-type (upper panels) and xpa-1 (lower panels) nematodes after exposures to 0 or 25 J/m² UVC irradiation either once on day 0 (single - S) or repeatedly for 3 days (chronic - C). Although untreated xpa-1 nematodes appeared slightly larger than wild-type, after 3 days of daily UVC exposure (C25), xpa-1 nematodes had a starved appearance and lack of embryos in the gonad. All images were taken at 100x magnification.

Figure 3. NER is required for normal growth in UVC-exposed adult C. elegans

Wild-type (left) and xpa-1 (right) adult growth after single (upper) or chronic (lower) exposures to 0, 6, 12, 25, 50, or 100 J/m² UVC irradiation. Mean size measurements (optical extinction) ± standard error of the mean for one representative experiment is presented. Optical extinction measurements of individual nematodes were performed on a COPAS Biosort.

Figure 4. NER is required for normal feeding in UVC-exposed adult C. elegans

Wild-type (left) and xpa-1 (right) adult feeding after single (upper) or chronic (lower) exposures to 0, 6, 12, 25, 50, or 100 J/m² UVC irradiation. Mean feeding measurements (red fluorescence) ± standard error of the mean for one representative experiment is presented. Red fluorescence measurements of individual nematodes were performed on a COPAS Biosort.

Figure 5. DNA damage accumulates in mutant C. elegans after chronic UVC exposures

Adult nematodes were exposed daily to 0, 12, 25, or 50 J/m², and frozen 24 h later (immediately prior to dosing of remaining nematodes) for QPCR-based DNA damage analysis. The first dose was at 24 h post-L4 molt (“day 0”). DNA damage measured at day 0 was in nematodes sampled immediately after exposure; on subsequent days, nematodes were sampled 24h after the previous exposure and immediately prior to the following dose. The effects of strain, dose, and day were all statistically significant, as were the strain by treatment and strain by day interactions (p < 0.0001 in all cases, 3-factor ANOVA). n = 4–8 per column; error bars represent standard error of the mean.

Figure 6. Global gene expression is comparable in the N2 and xpa-1 strains, but dramatically different in the glp-1 strain

The glp-1 samples cluster completely separately from the N2 and xpa-1 samples. The effect of year (and amplification/labeling protocol) is greater than the effect of UVC exposure within the glp-1 samples, and greater than either UVC exposure or strain for the N2 and xpa-1 strains. UVC exposure affects global gene expression more than strain for the N2 and xpa-1 strains. This heat map was generated using GeneSpring; hierarchical clustering was performed on conditions and genes (including all genes that passed basic quality filtering).

Figure 7. Expression of most DNA repair-related genes was not induced three hours after UVC exposure, and is in general low in germ cell-deficient nematodes

Many DNA repair genes are expressed at a lower level in glp-1 than N2 and xpa-1 young adults (all at 25°C, so that glp-1 nematodes lack germ cells). The only significantly UVC-induced DNA repair-related gene was pme-4 (shown in blue). See Supplemental data files 1 and 10 for exact strain-to-strain and UVC-induced fold-change values.

Figure 8. Inducible repair of UVC-induced DNA damage was not detected in young adult C. elegans

DNA lesions detected in glp-1 young adults either untreated or pre-treated with a single 6 J/m² UVC exposure, and then 18 h later exposed to a challenge dose of 0, 6, 12, 25, 50, or 100 J/m² as indicated. Nematodes were frozen at 0, 6, 12, 24, 48, and 72 h post-dosing. Note that the y-axes all indicate lesions/10kb but have different scales; the data are graphed to highlight relative rates of repair. The effects of dose, timepoint, and interactions of dose by timepoint and dose by pretreatment were all significant (p < 0.004 in all cases, 3-factor ANOVA); all other factors and interactions were not significant (p > 0.15 in all cases, 3-factor ANOVA). n = 4–8 per column; error bars represent standard error of the mean.