NER and HR pathways act sequentially to promote UV-C-induced germ cell apoptosis in Caenorhabditis elegans

Abstract

Ultraviolet (UV) radiation-induced DNA damage evokes a complex network of molecular responses, which culminate in DNA repair, cell cycle arrest and apoptosis. Here, we provide an in-depth characterization of the molecular pathway that mediates UV-C-induced apoptosis of meiotic germ cells in the nematode Caenorhabditis elegans. We show that UV-C-induced DNA lesions are not directly pro-apoptotic. Rather, they must first be recognized and processed by the nucleotide excision repair (NER) pathway. Our data suggest that NER pathway activity transforms some of these lesions into other types of DNA damage, which in turn are recognized and acted upon by the homologous recombination (HR) pathway. HR pathway activity is in turn required for the recruitment of the C. elegans homolog of the yeast Rad9-Hus1-Rad1 (9-1-1) complex and activation of downstream checkpoint kinases. Blocking either the NER or HR pathway abrogates checkpoint pathway activation and UV-C-induced apoptosis. Our results show that, following UV-C, multiple DNA repair pathways can cooperate to signal to the apoptotic machinery to eliminate potentially hazardous cells.

Figure 1

Several components of the HR repair pathway are required for UV-C-induced germ cell apoptosis. (a) mre-11 mutants fail to induce apoptosis in response to UV-C. Staged young adult mre-11(ok179) animals were treated with either 100 J/m² UV-C or 120 Gy X-rays and germ cell corpses were scored at the indicated time points. (b–e) Genetic characterization of rad-54 mutants. (b) rad-54(lf) results in increased levels of germ cell apoptosis. Germ cell corpses were scored every 12 h until 36 post the L4/adult molt in staged rad-54(ok615) or rad-54(tm1268) mutants, or rad-54(RNAi)-treated animals. (c–e) rad-54(lf)-induced germ cell death depends on spo-11 (c), cep-1 (d) and ATM/ATR function (e). (f) rad-54 mutants fail to induce apoptosis in response to UV-C and IR. Staged young adult rad-54(ok615) animals were treated with either 100 J/m² UV-C or 120 Gy X-rays and germ cell corpses were scored at the indicated time points. (g) Loss of spo-11 function does not restore UV-C- and IR-induced apoptosis in rad-54(RNAi) animals. Staged spo-11(ok79) L1 larvae were raised on bacteria expressing rad-54 or gfp dsRNA, and were treated with either 100 J/m² UV-C or 120 Gy X-rays as young adults. Germ cell corpses were scored at the indicated time points. (h) rad-51(lf) animals normally induce apoptosis in response to UV-C. Staged wild-type L1 larvae were raised on bacteria expressing rad-51 or control dsRNA, and were treated with either 100 J/m² UV-C or 120 Gy X-rays as young adults. Germ cell corpses were scored at the indicated time points. Data shown in all cases represent the average±S.D. of two or three independent experiments (n>20 animals for each experiment)

Figure 2

The HR pathway acts downstream of the NER pathway to induce UV-C-induced germ cell apoptosis. (a and c) Fluorescent microscopy of mid–late pachytene germ cells expressing RAD-54::YFP (a) or RPA-1::YFP (c). Germ cell nuclei from staged young adult wild-type animals were scored for the presence of YFP, 3 h after exposure to 100 J/m² of UV-C or 120 Gy of X-rays. RAD-54 or RPA-1 show a diffuse nuclear staining in control animals, but relocalize into distinct foci following treatment (arrowheads). (b) XPA-1, XPC-1 and MRE-11 are required for RAD-54 foci formation in response to UV-C, but not IR. (d and e) UV-C-induced RPA-1 foci require XPA-1 (d), but not MRE-11 (d) or RAD-54 (e). RAD-54::YFP and RPA-1::YFP foci were quantified as described in Materials and Methods. Data shown represent the average±S.D. of at least two experiments (n≥15 worms for each experiment)

Figure 3

C. elegans NER component XPG-1 is required for UV-C-induced germ cell apoptosis, but not for recruitment of RAD-54. Staged wild-type L1 larvae were raised on bacteria expressing xpb-1 (a), xpd-1 (b) or control dsRNA, and were treated with either 100 J/m² UV-C or 120 Gy X-rays as young adults. Germ cell corpses were scored at the indicated time points. We could not score the 36-h time point in these animals, as the germ lines started to degenerate. This is possibly owing to the fact that both XPB-1 and XPD-1 are components of TFIIH, and thus might also affect mRNA transcription. (c and d) Apoptosis was scored in xpg-1(tm1670) or xpg-1(tm1682) mutant animals after treatment with 100 J/m² UV-C (c) or 120 Gy X-rays (d). (e) XPG-1 is not required for UV-C-induced RAD-54 foci formation. RAD-54::YFP foci were quantified 3 h after treatment with 100 J/m² UV-C as described in Materials and Methods. (f) RAD-54, but not XPG-1, is required for UV-C-induced HUS-1 foci formation. HUS-1::GFP foci were quantified in mid–late pachytene germ cell nuclei from staged young adult animals expressing HUS-1::GFP (opIs34) 3 h after exposure to 100 J/m² of UV-C or 120 Gy of X-rays. Data shown in all cases represent the average±S.D. of three independent experiments (n>15 animals for each experiment). (g) Increased levels of apoptosis in rad-54 mutants are suppressed by xpg-1. Germ cell apoptosis was scored 12 and 24 h post the L4/adult molt in staged wild-type (wt), xpg-1(tm1670), rad-54(RNAi) and xpg-1(tm1670);rad-54(RNAi) animals. Data shown represent the average±S.D. of two independent experiments (n>20 animals for each experiment)

Figure 4

CPD repair kinetics in NER and HR pathway mutants. (a) Immunofluorescence images of germ cell nuclei from UV-C-irradiated wild-type, rad-54(ok615) and xpa-1(ok698) worms. Gonads were extruded and fixed immediately following UV-C radiation or after 14 h. Images in the upper panel represent the CPD staining (green), the nuclei DAPI staining (magenta) and an overlay of the CPD staining and DAPI in wild-type animals, resulting in a white ring where UV lesions are detected on germ cell chromatin. Although in the absence of NER in xpa-1(ok698) mutants nuclei remain white (lower panel), CPDs are removed in rad-54(ok615) mutants similar to wild type mutants (chromatin turns magenta). (b) CPD signal intensity after irradiation with 100 J/m² UV-C was determined by fluorescence image analysis of isolated gonads. Intensity before irradiation was defined as 0 and the value immediately after UV-C as 1 for each strain, and later time points were expressed as a fraction of the initial UV-C-induced intensity. At least 10 animals were scored per data point. Error bars represent the S.E.M. Repair kinetics for CPD lesions in transition cell nuclei of wild-type hermaphrodites (t_1/2 approx. 10 h) are in good agreement with repair kinetics reported for other systems. xpa-1(ok698) mutants lacking a functional NER show no significant repair, whereas rad-54(ok615) mutants exhibit a response similar to wild type mutants. (c) RAD-54::YFP foci develop slowly and persist for long in UV-C-irradiated wild-type animals. Mid–late pachytene germ cell nuclei from staged young adult wild-type animals were scored for the presence of RAD-54::YFP foci, at 5, 10, 20 min, 1, 2, 4, 8 and 16 h post 100 J/m² of UV-C treatment

Figure 5

UV-C-induced apoptosis requires the action of both the NER and HR pathways. (a) Summary of the genetic requirements for the recruitment of NER and HR pathway components onto DNA lesions, as well as the induction of apoptosis following UV-C treatment. (b) Molecular model for UV-C-induced apoptosis in C. elegans. The NER machinery (blue) transforms a fraction of the UV-C lesions into other types of DNA damage (e.g., double-strand breaks). These, in turn, are then recognized by the HR machinery (green), leading to DNA repair and/or apoptosis of the damaged cell. Remarkably, XPG-1 defines a new branch that acts downstream of 9-1-1 to induce apoptosis