There have been mixed reports on the effects of mutations in the xpa-1
gene on lifespan in C. elegans
strains. Hyun et al. [17
] recently reported a markedly shorter lifespan both for the ok698
deletion allele of xpa-1
(used in the current report) and for RNAi-based knockdown of XPA-1. On the other hand, previous reports using both xpa-1
) and rad-3(mn157)
, a strain carrying an independently isolated deletion mutation of xpa-1
], detected no difference [18
]. In these experiments, we detected no difference in lifespan under control conditions.
However, our results are in agreement with those of Hyun et al. [17
] with respect to the dramatic decrease in lifespan in xpa-1
nematodes following adult UVC exposure. We further demonstrate that adult growth and feeding are exquisitely sensitive to UVC damage in the context of NER deficiency, and that these phenotypes are associated with a dramatic increase in DNA damage accumulation. Our lowest dose of UVC (6 J/m2
), led to as much or more reduction in lifespan, growth, and feeding in xpa-1
nematodes as did our highest dose (100 J/m2
) in wild-type animals. The lack of growth in adults could be due to decreased feeding resulting from a loss in transcriptional competence [19
], or DNA damage-mediated inhibition of endoreduplication upon which adult growth is dependent [41
]. We found that feeding was strongly inhibited in UVC-exposed xpa-1
young adults, although it cannot have been completely abolished since starved young adults do not survive past 3 days.
Even using microarray detection of global gene expression, we detected very few differences between the wild-type and xpa-1
strains. K07G5.3, which is located immediately adjacent to xpa-1
on chromosome I, was the gene that was most convincingly differentially expressed, and seems most likely to reflect dysregulation due to the xpa-1
mutation. Similarly, while there was a clear transcriptional response to UVC in all strains, there were also few differences between wild-type and xpa-1
nematodes in that response, suggesting that immediate damage-induced signaling is not dramatically altered in xpa-1
nematodes. We deliberately chose an early time point to avoid detecting transcriptomic differences resulting from the greater persistence of DNA damage in xpa-1
nematodes; a significant amount of UVC-induced DNA damage persists at three hours even in NER-proficient nematodes [23
]. These results suggest that the dramatic gene-environment interactions observed in xpa-1
nematodes can be attributed to the persistence of DNA damage in this strain, rather than to differential signaling mediated by xpa-1
under control conditions. It is also possible, however, that the UVC-induced xpa-1
phenotypes result from altered signaling at a different time point, or non-transcriptionally-mediated signaling.
Our results show that not just lifespan, but adult growth, feeding, heat shock resistance, egg production, and even global gene expression in xpa-1 nematodes are nearly identical to wild-type under control conditions. Thus, the level of background DNA damage that occurs in nematodes grown under our laboratory conditions is low enough that no phenotype could be detected using these measures. This suggests that the selective pressure that maintains NER in this short-lived organism must result from genotoxic stressors not encountered under controlled laboratory conditions, or require multiple generations to be evident.
The inducibility of DNA repair is important to characterize in C. elegans
given the increasing use of this species for studies of DNA damage and repair. NER is inducible in bacteria and yeast [42
], but inducibility of NER proteins or repair kinetics appears to be less robust and possibly species-specific in higher eukaryotes [48
]. This raises the important question of whether C. elegans
, a simple eukaryote, is more like the very simple eukaryote yeast, or more like the higher eukaryotes for which it is often used as a model, with respect to DNA damage inducibility. We detected the induction of very few DNA repair genes, and in fact very few DNA damage-responsive genes in general three hours post-UVC exposure, although DNA repair genes are induced by 1–3h in yeast [50
]. This lack of induction of DNA repair genes was observed both in germ cell- and embryo-containing young adults (wild-type and xpa-1
strains) and in young adults composed entirely of terminally differentiated somatic cells (glp-1
strain). Consistent with these results, Greiss et al. [52
] also detected induction of almost no DNA repair-related genes in C. elegans
exposed to ionizing radiation. Furthermore, although induction of some DNA repair genes is p53-dependent in mammals, Derry et al. [53
] detected no genes that were UVC-induced in a cep-1
Since it is possible that our microarray experiment missed an important time point for the detection of transcriptional regulation, or that NER might be inducible through non-transcriptional mechanisms, we also directly tested repair kinetics, but observed no induction. There are caveats associated with this observation. First, we measured repair in both strands of a transcribed gene, rather than specific repair of either transcribed or non-transcribed regions of the genome. Thus, if only transcription-coupled or global genomic repair were induced [48
], it is possible that such a signal would be undetectable. Second, we did not measure repair in germ cells or early embryos, which have much higher levels of DNA repair gene expression (this manuscript and [23
]). The question of whether DNA repair is faster and/or more inducible at earlier lifestages is an important one that should be addressed in future work. Finally, it is conceivable that a different pre-exposure dose might be more effective.
The lifestage-dependent difference in expression of DNA repair as well as other DNA damage-responsive genes is dramatic. Transcription of a high proportion of the components of the DNA damage response is much lower in glp-1
than in gravid wild-type or xpa-1
young adults, despite the dilution of the germ cells/early embryos by somatic cells. Interestingly, one exception to this pattern was genes involved in nonhomologous end-joining, of which only one was higher in germ cell-containing nematodes (cku-80
, ~3.5-fold higher). This is consistent with a previous study that found that nonhomologous end-joining is important in non-cycling somatic cells [13
]. While our experiments cannot distinguish between germs cells and early embryos, such experiments would be interesting given the fact that cell cycle checkpoint activation is silenced in C. elegans
in early embryos, but not in the germline [12
]. Importantly, we previously showed that there was no detectable difference in UVC damage repair kinetics between wild-type and glp-1
nematodes when measured in starved L1s, prior to germ cell proliferation [23
It would appear that under normal conditions, DNA damage-responsive proteins are present at sufficient levels in adult somatic cells to carry out their functions, as evidenced by the fact that UVC damage is repaired in adults [23
], and checkpoint proteins function in adult somatic cells [54
]. However, it is possible that levels become insufficient with time, as suggested by our observation of decreased repair in older adults [23
]. This insufficiency might be exacerbated by stressful environmental conditions (e.g., exposure to genotoxins), and is particularly interesting in the context of the observation by Weidhaas et al.
] that checkpoint proteins, best known for conferring susceptibility to DNA damage-induced apoptotic cell death in germ cells, also confer resistance to DNA damage-induced necrotic cell death in somatic cells.