A new thermosensitive smc-3 allele reveals involvement of cohesin in homologous recombination in C. elegans

Abstract

The cohesin complex is required for the cohesion of sister chromatids and for correct segregation during mitosis and meiosis. Crossover recombination, together with cohesion, is essential for the disjunction of homologous chromosomes during the first meiotic division. Cohesin has been implicated in facilitating recombinational repair of DNA lesions via the sister chromatid. Here, we made use of a new temperature-sensitive mutation in the Caenorhabditis elegans SMC-3 protein to study the role of cohesin in the repair of DNA double-strand breaks (DSBs) and hence in meiotic crossing over. We report that attenuation of cohesin was associated with extensive SPO-11-dependent chromosome fragmentation, which is representative of unrepaired DSBs. We also found that attenuated cohesin likely increased the number of DSBs and eliminated the need of MRE-11 and RAD-50 for DSB formation in C. elegans, which suggests a role for the MRN complex in making cohesin-loaded chromatin susceptible to meiotic DSBs. Notably, in spite of largely intact sister chromatid cohesion, backup DSB repair via the sister chromatid was mostly impaired. We also found that weakened cohesins affected mitotic repair of DSBs by homologous recombination, whereas NHEJ repair was not affected. Our data suggest that recombinational DNA repair makes higher demands on cohesins than does chromosome segregation.

Figure 1. The new allele smc-3 (t2553).

(A) Domain organization of C. elegans SMC-3 highlighting the position of the L1081F amino acid change; domain organizations predicted by CDART . (B) Alignment of SMC-3 sequences for the indicated organisms highlights conservation of the mutated leucine (highlighted in red in t2553).

Figure 2. Fragmentation at diakinesis resulting from defective DSBs repair.

(A) Representative DAPI structures found at diakinesis in: (i) top: wild type, bottom: smc-3; (ii) top: spo-11, bottom: smc-3; spo-11; (iii) top: mre-11, bottom: smc-3;mre-11; (iv) top: him-3, bottom: smc-3;him-3; and (v) top: him-17, bottom: smc-3;him-17. (B) Quantification of DNA fragmentation at last diakinesis. Classes (fragments, univalents, bivalents, and DNA masses) were defined by the volume (v) and the “sphericity” (s) of the DAPI structures at the last diakinesis before the spermatheca (see Materials and Methods). Single star indicates significant differences (p<0.05, Student's t-test) for fragments, univalents, bivalents, and masses between double mutants and the smc-3 single mutant. Error bars represent SD.

Figure 3. Chromosomes axes are impaired in smc-3.

(A) Immunostaining of SMC-3 in wild-type and smc-3 worms. (B) Squashed nuclei untreated and washed with sarkosyl in wild-type and smc-3 worms in the TZ and pachytene stained with anti-REC-8 antibody. (C) Squashed nuclei washed with sarkosyl stained with anti-HTP-3 in wild-type and smc-3 worms in TZ and pachytene. Bar: 10 µm.

Figure 4. smc-3 mutants are defective in repair of meiotic DSBs.

Left, immunostaining of RAD-51. Right, quantification of RAD-51 foci in wild-type (A), smc-3 (tm2553) (B), smc-3;spo-11 (C), smc-3;mre-11 (D). and smc-3;spo-11; mre-11 (E) worms. Gonads were divided into six zones of equal length for quantification. Arrowheads indicate the zone with clustered chromatin. Bar: 10 µm.

Figure 5. The t2553 allele impairs mitotic HR repair but not NHEJ repair.

(A) Schematic drawing of an embryo with germ (bold circle) and somatic cells (circle). Green color highlights cells repairing DSBs by NHEJ; red cells repair via HR. In the adult, small red circles represent spermatozoa and open ovals represent embryos. (B) Percentage of arrested L4 66 h after 60 Gy irradiation of late-stage embryos with the indicated genotypes. Error bars represent standard error of the mean (SEM). (C) DAPI staining of wild-type and smc-3 gonads released from worms irradiated (60 Gy) as late-stage embryos. Stars mark the distal tip of the gonad. Bar: 10 µm.

Figure 6. DNA damage checkpoints are working in smc-3.

Proportion of nuclei with “normal” (<3.85 µm) or “double” (≥3.85 µm) diameters before and 8 h after 60 Gy irradiation (more than 200 nuclei assayed in five different gonads per genotype for the indicated genotypes (N2, smc-3, and mrt-2). Error bars represent SEM. (B) Percentage of surviving worms 48 h after 60 Gy irradiation. Error bars represent SEM. More than 500 L1s were scored per genotype.