Meiotic Double-Strand Break Proteins Influence Repair Pathway Utilization

Abstract

Double-strand breaks (DSBs) are among the most deleterious lesions DNA can endure. Yet, DSBs are programmed at the onset of meiosis, and are required to facilitate appropriate reduction of ploidy in daughter cells. Repair of these breaks is tightly controlled to favor homologous recombination (HR)-the only repair pathway that can form crossovers. However, little is known about how the activities of alternative repair pathways are regulated at these stages. We discovered an unexpected synthetic interaction between the DSB machinery and strand-exchange proteins. Depleting the Caenorhabditis elegans DSB-promoting factors HIM-5 and DSB-2 suppresses the formation of chromosome fusions that arise in the absence of RAD-51 or other strand-exchange mediators. Our investigations reveal that nonhomologous and theta-mediated end joining (c-NHEJ and TMEJ, respectively) and single strand annealing (SSA) function redundantly to repair DSBs when HR is compromised, and that HIM-5 influences the utilization of TMEJ and SSA.

Keywords: C. elegans; DNA repair; WormBase; double-strand break; meiosis; pathway choice.

Figure 1

The rad-51;him-5 double mutant phenocopies spo-11. (A–E) The rad-51;him-5 double mutant shows a phenotype similar to spo-11 worms with 12 univalent-like structures in diakinesis nuclei. Pictures show representative images of DAPI-stained diakinesis nuclei of wild type and mutants at day 2 of adulthood. Bar, 2 µm. (A) Wild type (n = 45); (B) spo-11(me44) (n = 26); (C) him-5(e1490) (n = 66); (D) rad-51(lg8701) (n = 40), (E) rad-51(lg8701);him-5(e1490) (n = 33). (F) Quantification of DAPI-bodies in diakinesis nuclei. Magenta bars demarcate the median and interquartile range.

Figure 2

Depletion of him-5 suppresses chromosome fusions that arise in multiple early HR-defective mutants. (A) rad-54 and (B) mre-11S mutant animals exhibit chromosomal clumping that is suppressed by the depletion of him-5. (C and D) Irradiation of rad-51;him-5 does not restore a rad-51-like phenotype when him-5 is absent (C) rad-51 + IR control; (D) rad-51; him-5(e1490). DAPI-stained diakinesis nuclei at day 1 (A and B) or day 2 (C and D) of adulthood. Bar, 2 µm. (E) Quantification of DAPI-bodies in diakinesis nuclei of irradiated (+IR; blue dots) rad-51(lg8701) (n = 45), rad-51(lg8701);him-5(e1490) (n = 71), and nonirradiated (clear dots) rad-51(lg8701);him-5(e1490) (n = 33). Magenta bars indicate the median and the interquartile range. P-values calculated with two-tailed Mann-Whitney tests.

Figure 3

SPO-11-mediated DSBs are catalyzed in rad-51;him-5. (A) DNA fragments are not observed in the control spo-11,rec-8 strain. (B) The presence of DSBs catalyzed by SPO-11 in rad-51;him-5 is revealed by the presence of chromatin fragments in rad-51,rec-8;him-5. Representative images of DAPI-stained diakinesis nuclei in 1-day-old adults. Arrows point to fragments of chromatin. Bar, 2 µm. (C) Quantification of DAPI-bodies in diakinesis nuclei: rec-8(ok978) (n = 16), spo-11(me44),rec-8(ok978) (A; n = 44), and rad-51(lg8701),rec-8(ok978);him-5(e1490) (B; n = 35). Diamonds indicate nuclei containing at least one fragment of chromatin. Magenta bars show the median and the interquartile range.

Figure 4

c-NHEJ, TMEJ and SSA contribute to DSB repair in the HR defective rad-51 mutant. (A–D, F, and G) Representative images and (E) Quantification of DAPI-stained diakinesis nuclei from 1-day-old adults of designated genotypes. Bar, 2 µm. Magenta bars indicate the median and the interquartile range. P-values are calculated with Kruskal-Wallis multicomparison tests (n.s.: not significant, P > 0.19). rad-51(lg8701) (n = 106); cku-70(tm1524);rad-51(lg8701) (n = 53); polq-1(tm2026);rad-51(lg8701) (n = 43); xpf-1(e1487);rad-51(lg8701) (n = 40); polq-1(tm2026),cku-70(tm1524);rad-51(lg8701) (n = 60); xpf-1(e1487);polq-1(tm2026),cku-70(tm1524);rad-51(lg8701) (n = 108).

Figure 5

c-NHEJ is the major repair pathway for IR-induced breaks in rad-51;him-5 mutants. (A–F). Representative images and (G) Quantification of DAPI-stained diakinesis nuclei of 2-day-old, irradiated (+IR) adults. Bar, 2 µm. (G) Magenta bars indicate the median and the interquartile range. Kruskal-Wallis multiple comparisons to rad-51;him-5 (n.s.: not significant, P > 0.05). rad-51(lg8701);him-5(e1490) (n = 71), cku-80(ok851);rad-51(lg8701);him-5(e1490) (n = 80), polq-1(tm2027);rad-51(lg8701);him-5(e1490) (n = 67), xpf-1(e1487);rad-51(lg8701);him-5(e1490) (n = 61), polq-1(tm2026),cku-70(tm1524);rad-51(lg8701);him-5(e1490) (n = 63), xpf-1(e1487); polq-1(tm2026);rad-51(lg8701);him-5(e1490) (n = 64), xpf-1(e1487); polq-1(tm2026),cku-70(tm1524);rad-51(lg8701);him-5(e1490) (n = 59).

Figure 6

DSB repair is regulated differently in rad-51 and mre-11S mutants. (A–C) IR-induced breaks do not change the phenotype of rad-51 mutants, while they do affect mre-11S mutants. Shown are DAPI-bodies in representative diakinesis nuclei of unirradiated and irradiated (+IR) animals of indicated genotypes. (D–F) Depleting mre-11S or exo-1 in rad-51 mutants affects chromatin morphology, suggesting alterations in how DSBs are repaired. (A, B, D, and E) Images of diakinesis nuclei of 2-day-old adults of indicated genotypes. (C and F) Quantification of DAPI-stained bodies in diakinesis nuclei. (G–N) him-5 does not influence resection, but MRE-11 and EXO-1 act in concert for the repair of IR-induced breaks. (G–J) mre-11S or exo-1 mutations have no effect on diakinesis chromosome morphology in the rad-51;him-5 background. (K–N) By contrast, mre-11S and exo-1 affect repair of IR-induced damage, independently of him-5. (G–I and K–M) Pictures show DAPI-stained diakinesis nuclei of adults on day 2 of adulthood. Bar, 2 µm. (J and K) Quantification of DAPI-bodies in diakinesis nuclei of indicated genotypes. Magenta bars indicate median and interquartile range. P-values calculated from Kruskal Wallis tests (n.s.: not significant, P > 0.15). rad-51(lg8701) (n = 40), mre-11S(iow1) (n = 30), rad-51(lg8701)+IR (n = 39), mre-11S(iow1)+IR (n = 47). (F) rad-51(lg8701) (n = 106), rad-51(lg8701);mre-11S(iow1) (n = 50), exo-1(tm1842);rad-51(lg8701) (n = 63). (J) rad-51(lg8701);him-5(e1490) (n = 33), rad-51(lg8701);mre-11S(iow1),him-5(e1490) (n = 57), exo-1(tm1842);rad-51(lg8701);him-5(e1490) (n = 28), exo-1(tm1842);rad-51(lg8701);mre-11S(iow1),him-5(e1490) (n = 50). (N) rad-51(lg8701);him-5(e1490)+IR (n = 71), rad-51(lg8701);mre-11S(iow1),him-5(e1490)+IR (n = 60), exo-1(tm1842);rad-51(lg8701);him-5(e1490)+IR (n = 39), exo-1(tm1842);rad-51(lg8701);mre-11S(iow1),him-5(e1490)+IR (n = 101).

Figure 7

Loss of dsb-2 function impacts repair in rad-51 mutants, similar to him-5. (A and B) As described (Rosu et al. 2013), IR suppresses the univalent phenotype induced by loss of dsb-2, suggesting the gene is required for DSB induction. (A) Quantification of diakinesis oocytes of indicated genotypes. Wild type (WT, n = 45), dsb-2(me96) (n = 30), WT+IR (n = 52), dsb-2(me96) +IR (n = 44). Two-tailed Mann-Whitney tests are indicated on top of the graph as exact P-values (n.s.: not significant, P = 0.5567). (B) Representative images of diakinesis oocytes in irradiated 2-day-old, dsb-2 mutant animals. (C–E) Depletion of dsb-2 suppresses fusions in rad-51 mutants. (C–E) Diakinesis nuclei in dsb-2;rad-51 double mutants contain 12 intact univalents that are largely unaffected by IR-induced breaks. (C) Quantification and (D and E) Representative images of DAPI-stained diakinesis nuclei. rad-51(lg8701) (n = 40), dsb-2(me96);rad-51(lg8701) (n = 14), rad-51 +IR (n = 45), dsb-2;rad-51(lg8701) +IR (n = 39). two-tailed Mann-Whitney test (n.s.: not significant, P > 0.16). (F and G) SPO-11-mediated breaks are formed in dsb-2;rad-51 as seen by (F) chromatin fragments in the nuclei of dsb-2;rad-51,rec-8 (compare spo-11,rec8 in Figure 3A). (G) Quantification of DAPI bodies in diakinesis oocytes of spo-11(me44),rec-8(ok978) (n = 44), dsb-2(me96);rad-51(lg8701),rec-8(ok978) (n = 23). Diamonds correspond to nuclei containing at least one fragment of chromatin. Scale bars in images = 2 µm. Magenta bars in dot plots indicate median and interquartile range.