Developmental modulation of nonhomologous end joining in Caenorhabditis elegans

Abstract

Homologous recombination and nonhomologous end joining (NHEJ) are important DNA double-strand break repair pathways in many organisms. C. elegans strains harboring mutations in the cku-70, cku-80, or lig-4 NHEJ genes displayed multiple developmental abnormalities in response to radiation-induced DNA damage in noncycling somatic cells. These phenotypes did not result from S-phase, DNA damage, or mitotic checkpoints, apoptosis, or stress response pathways that regulate dauer formation. However, an additional defect in him-10, a kinetochore component, synergized with NHEJ mutations for the radiation-induced developmental phenotypes, suggesting that they may be triggered by mis-segregation of chromosome fragments. Although NHEJ was an important DNA repair pathway for noncycling somatic cells in C. elegans, homologous recombination was used to repair radiation-induced DNA damage in cycling somatic cells and in germ cells at all times. Noncycling germ cells that depended on homologous recombination underwent cell cycle arrest in G2, whereas noncycling somatic cells that depended on NHEJ arrested in G1, suggesting that cell cycle phase may modulate DNA repair during development. We conclude that error-prone NHEJ plays little or no role in DNA repair in C. elegans germ cells, possibly ensuring homology-based double-strand break repair and transmission of a stable genome from one generation to the next.

Figure 1.—

Deletions of cku-70, cku-80, and lig-4 remove essential regions of these NHEJ proteins. Gene structures are shown for (A) cku-70, (B) cku-80, and (C) lig-4 (NCBI accession nos. CAB55094, CAA83623, and AAK85439, respectively). Predicted protein structural domains are shown above each gene model. Shaded bars below each gene model indicate positions of deletion mutations. Introns >1000 bp are not connected.

Figure 2.—

Stages of C. elegans development used in this study. (A) Embryonic and larval stages (not to scale). Germ or somatic cells are depicted in shaded or open circles, respectively, sperm are tiny solid spots, and embryos are open ovals. (B) Cell proliferation occurs in early- but not late-stage C. elegans embryos.

Figure 3.—

Radiation response of NHEJ mutant germlines is normal. (A) NHEJ mutations do not significantly affect the survival of progeny of gamma-irradiated L4 larvae. (B) DAPI-stained oocyte nuclei (indicated by dashed circles) of adults derived from irradiated L4 larvae reveal a normally condensed complement of six bivalents for N2 wild-type and cku-80 mutants but broken chromosomes for mrt-2 strains. (C) NHEJ mutations such as cku-80(ok861) do not confer a role in germline DSB repair in the absence of the hus-1 DNA damage response gene. Progeny of 10 worms from each strain were scored for viability as described (Ahmed et al. 2001), which is a sensitive measure of genome instability in the C. elegans germline. (D) Arrested NHEJ mutant germ cells are not deficient for DSB repair. Progeny of adults derived from late-stage embryos irradiated with 50 Gy were scored for embryonic lethality. Error bars correspond to standard deviations. DAPI-stained germline nuclei of adults derived from late-stage embryos of (E) N2 wild type, (F) cku-80, or (G) mrt-2 strains irradiated with 70 Gy. Pachytene nuclei at the border of the transition zone are shown.

Figure 4.—

Somatic functions for HR and NHEJ in early or late embryogenesis, respectively. (A) rrf-3 control or rrf-3;lig-4(ok716) C. elegans strains were fed on bacteria expressing double-stranded RNA for vector or for rad-51, mre-11, rad-50, or rad-54 HR genes, and early stage F₂ embryos derived from F₁ progeny were irradiated at 30 Gy. Survival was computed relative to unirradiated controls as the ratio of live worms to total worms in irradiated samples divided by the ratio of live worms to total worms in the unirradiated samples. Averages for two experiments are shown. Late Egg Rad phenotypes of NHEJ mutants are shown as (B) the Gro phenotype; (C) combined totals for Unc, Rup, and Pvl phenotypes; and (D) individual totals for Pvl, Vul, Egl, Rup, and Unc phenotypes. For B, developmental stages were scored by monitoring embryos and larvae every 2 hr, from the time of irradiation until adulthood. Larval stages were defined as initiating when >90% of the worms had reached a stage. Results in C are shown for representative experiments that were repeated at least twice for cku-80(ok861) and lig-4(ok716) with similar results. (D) N2 wild type, cku-80(ok861), lig-4(ok716), cku-80(tm1203), lig-4(tm750), and cku-70(ok1524) were irradiated at 90 Gy and scored as described in materials and methods (n = 91, 96, 101, 116, and 87, respectively). Note that the Rup phenotype was larval lethal and that the percentages of Pvl, Vul, and Egl phenotypes were derived exclusively from animals that survived until adulthood. rad-51(RNAi) or mre-11(RNAi) strains did not display Late Egg Rad phenotypes. (E) Deficiency for HR genes does not cause a Slow Growth phenotype in irradiated late-stage embryos, nor does it suppress the Slow Growth phenotype of NHEJ mutants such as lig-4(ok716). Experiments were conducted in rrf-3 or rrf-3;lig-4 genetic backgrounds, and the percentage of larvae that had reached the L4 stage was scored at 48 hr post-irradiation. The effectiveness of the HR gene RNAi was confirmed by observing complete sterility for irradiated late-stage embryos. n = 100 scored for each strain, except for mre-11(RNAi) (n = 61 at 0 Gy, n = 64 at 90 Gy), rad-54(RNAi);lig-4 (n = 95 at 0 Gy, n = 84 at 90), and rad-50(RNAi);lig-4 (n = 62 at 90 Gy). Error bars correspond to standard deviations.

Figure 5.—

Phenotypes of late-stage embryos irradiated with 70 Gy IR. (A) Adult phenotypes. Arrows indicate gonad protruding from ruptured vulva (Rup) or prodtruding vulva (Pvl). (B) Irradiated late-stage embryos of NHEJ mutants can give rise to thin larvae in comparison to an irradiated N2 wild-type control. An unirradiated daf-2(m41) dauer larva is shown for comparison.

Figure 6.—

Irradiated NHEJ mutant dauers exhibit some phenotypes of irradiated late-stage NHEJ mutant embryos. The mutations were cku-80(ok861), lig-4(ok716), daf-8, and daf-2(m41). Similar results were obtained with daf-2(e1368). Dauer larvae were daf-8 (n = 21 and 25), lig-4;daf-8 (n = 46 and 21), cku-80;daf-8 (n = 23), daf-2 (n = 55 and 30), and daf-2;cku-80 (n = 48 and 30). L3 larvae were daf-8 (n = 30), daf-8;lig-4 (n = 47 and 56), daf-8;cku-80 (n = 42), daf-2 (n = 30 and 33), and daf-2;cku-80 (n = 59). Results were averaged from two experiments and error bars correspond to standard deviations.

Figure 7.—

Serotonin does not rescue the egg-laying defect of irradiated NHEJ mutants. An egl-8 mutant strain, which has an Egl phenotype as a consequence of a neurotransmission defect and is rescued by serotonin (Bastiani et al. 2003), is shown as a control. Ten gravid adults from each sample were placed in individual wells containing 7.5 mm serotonin (5-hydroxytryptamine; Sigma, St. Louis) for 90 min and eggs were counted as described (Bastiani et al. 2003).

Figure 8.—

Karyokinesis failure of intestinal nuclei in larvae derived from late-stage NHEJ mutant embryos. (A) Example of an elongated intestinal nucleus observed in a DAPI-stained L2 larva derived from an irradiated lig-4(ok716) mutant late-stage embryo. Several normal polyploid intestinal nuclei are observed as large, round halos, whereas an elongated intestinal nucleus is also observed (arrow). A string of diploid ventral nerve cord nuclei are observed on the left side of the larva. (B) Quantification of karyokinesis defects in N2 wild-type, cku-80(tm1203), or lig-4(ok716) strains. The number of elongated nuclei per DAPI-stained adult derived from late-stage embryos is indicated (n = 20/strain, with each experiment repeated twice). Mean scores are shown and error bars represent standard deviations. (C) An elt-2∷GFP reporter labels intestinal nuclei, which normally complete nuclear division and clearly separate in L2 larvae derived from irradiated late-stage embryos, as shown. (D and E) In a cku-80(tm1203);elt-2∷GFP strain, intestinal nuclei with karyokinesis defects (arrows) are observed after the L1/L2 molt in larvae derived from irradiated late-stage embryos.

Figure 9.—

Synergy of NHEJ mutations with him-10 at 30 Gy. (A) The Gro phenotype was scored as the percentage of larvae that had reached the L4 stage 48 hr after their embryos were laid (n = 64 and 72 for cku-80; n = 53 and 70 for him-10; n = 36 and 70 for cku-80;him-10). (B) Other Late Egg Rad phenotypes were scored for the same strains (n = 50/strain/experiment). Egl worms were not observed either in him-10;cku-80 double mutants or in him-10 single mutants, because radiation-induced mitotic and meiotic germline defects that occur as a consequence of the him-10 mutation result in complete sterility. Experiments were repeated twice in each case and error bars correspond to standard deviations. At doses >50 Gy, for which wild type was unaffected, the him-10 mutant exhibited Late Egg Rad phenotypes other than Gro, although their penetrance was low in comparison with either NHEJ mutants or him-10;NHEJ double mutants (data not shown).