Synapsis-defective mutants reveal a correlation between chromosome conformation and the mode of double-strand break repair during Caenorhabditis elegans meiosis

Abstract

SYP-3 is a new structural component of the synaptonemal complex (SC) required for the regulation of chromosome synapsis. Both chromosome morphogenesis and nuclear organization are altered throughout the germlines of syp-3 mutants. Here, our analysis of syp-3 mutants provides insights into the relationship between chromosome conformation and the repair of meiotic double-strand breaks (DSBs). Although crossover recombination is severely reduced in syp-3 mutants, the production of viable offspring accompanied by the disappearance of RAD-51 foci suggests that DSBs are being repaired in these synapsis-defective mutants. Our studies indicate that once interhomolog recombination is impaired, both intersister recombination and nonhomologous end-joining pathways may contribute to repair during germline meiosis. Moreover, our studies suggest that the conformation of chromosomes may influence the mode of DSB repair employed during meiosis.

Figure 1.—

Quantitative time-course analysis of RAD-51 foci during meiotic prophase. Germline diagram indicates the zones along the germline throughout which RAD-51 foci were scored for all nuclei. Zone 1 and most of zone 2 consist of nuclei undergoing mitotic divisions (premeiotic tip). Zone 3 consists of nuclei entering meiosis (transition zone). Zones 4–7 consist of nuclei in early through late pachytene. Levels of observed RAD-51 foci are indicated by the color code. Histograms depict the quantitation of RAD-51 foci in germlines of the indicated genotypes. Positions along the x-axis correspond to the zones along the germline as indicated in the diagram. The y-axis indicates the percentage of nuclei falling into each corresponding category indicated by the color code.

Figure 2.—

Sister chromatid-mediated recombination and NHEJ are alternative modes of meiotic DSB repair in syp-3 mutants. Elevated levels of RAD-51 foci are apparent in syp-3(ok758) and further accentuated if sister chromatid cohesion is disrupted in syp-3(ok758); rec-8(RNAi). In syp-3(me42), levels of RAD-51 foci are comparable to wild-type, lig-4, or syp-3(me42); lig-4. However, they are elevated in syp-3(me42); lig-4; rec-8(RNAi) and in syp-3(me42); syp-2. Images are projections halfway through 3D data stacks of mid-pachytene (zone 5) nuclei. DAPI-stained chromosomes (blue); α-RAD-51 (red). Bars, 2 μm.

Figure 3.—

Examining how changes in chromosome structure affect DSB repair. DAPI-stained oocytes at late diakinesis (projections encompass entire nuclei) are depicted. rec-8(RNAi) results in 24 separated sister chromatids with occasional fragments (indicated by arrowhead and enlarged in inset). In syp-3(ok758); rec-8(RNAi) and syp-3(me42); rec-8(RNAi), more frequent chromosome fragmentation is observed, as indicated by arrows (arrowhead indicates example depicted at a higher magnification in the inset). In addition, chromosome aggregation is observed in syp-3(ok758); rec-8(RNAi). Bars, 2 μm.

Figure 4.—

Uncoupling the exit from a clustered organization from meiotic DSB formation. DAPI-stained mid-pachytene nuclei (projections are halfway through 3D data stacks). syp-3(me42); spo-11 and syp-3(ok758); spo-11 are indistinguishable from the corresponding syp-3 single mutants. Bars, 2 μm. Histograms depict the quantitation of RAD-51 foci in germlines of the indicated genotypes (as described in Figure 1). The average number of nuclei scored per zone (n) for spo-11, syp-3(me42); spo-11, syp-3(ok758); spo-11 were: zone 1 (n = 138), zone 2 (n = 158), zone 3 (n = 145), zone 4 (n = 138), zone 5 (n = 127), zone 6 (n = 102), and zone 7 (n = 90).