Robust crossover assurance and regulated interhomolog access maintain meiotic crossover number

Abstract

Most organisms rely on interhomolog crossovers (COs) to ensure proper meiotic chromosome segregation but make few COs per chromosome pair. By monitoring repair events at a defined double-strand break (DSB) site during Caenorhabditis elegans meiosis, we reveal mechanisms that ensure formation of the obligate CO while limiting CO number. We find that CO is the preferred DSB repair outcome in the absence of inhibitory effects of other (nascent) recombination events. Thus, a single DSB per chromosome pair is largely sufficient to ensure CO formation. Further, we show that access to the homolog as a repair template is regulated, shutting down simultaneously for both CO and noncrossover (NCO) pathways. We propose that regulation of interhomolog access limits CO number and contributes to CO interference.

Fig. 1

Meiotic recombination initiated by Mos1 excision-induced DSBs. (A) Mos1 excision-induced DSB repair assay. (B) Time-course analysis of IH recombination induced at the Mos1 site. Chart shows the incidence of unc-5(+) recombinants among progeny derived from eggs laid in the indicated time intervals (hours phs) and the incidence of CO and NCO repair types (8) (table S3).

Fig. 2

COs induced by Mos1 excision inhibit formation of other COs on the same chromosome. Modified Mos1 DSB repair assay to evaluate CO interference. Purple indicates N2-derived chromosome IV segments, orange indicates segments derived from the Hawaiian strain background, and ticks indicate genetic positions of SNP markers. Genotypic classes (and number of occurrences) are shown for chromosomes that had undergone either a CO or NCO event at Mos1.

Fig. 3

Mos1-induced DSBs are efficiently converted into COs when endogenous DSBs are absent. (A) IH recombinants and CO versus NCO repair types after DSB induction at the Mos1 site in spo-11 worms. Here and in Fig. 4, a subset of recombinants could not be scored for CO versus NCO repair type because of premature death or aneuploidy (8). (B) Mos1-initiated COs in the spo-11 background result in cytologically detectable chiasmata. (Top) The full complement of chromosomes in a single diakinesis-stage oocyte nucleus. (Left) WT nucleus with six pairs of homologs connected by chiasmata; four are oriented so that chromosome axis protein HIM-3 is visible in a cross-shaped structure marking the chiasma. (Center and right) Nuclei from a spo-11 mutant carrying unc-5(Mos1), 40 hours after heat-shock induction of transposase; the center nucleus has 12 unattached chromosomes (univalents), whereas the left nucleus contains one bivalent with a Mos1-induced chiasma (arrowhead). (Bottom) Single focal plane highlighting the chiasma and a nearby univalent. (C) Quantitation of chiasma formation elicited by heat shock–induced DSBs; parentheses indicate numbers of nuclei scored. Detection of nuclei with unambiguous chiasmata after DSB induction is highly significant, because these were never detected in controls (*P = 0.001). The 11 DAPI bodies (ambiguous) class can include both (i) nuclei with two univalents in close proximity that are difficult to resolve and (ii) nuclei with a real CO/chiasma in which the chiasma is in an unclear orientation. DAPI, 4′,6-diamidino-2-phenylindole.

Fig. 4

Time-course analysis of Mos1-induced meiotic IH recombination in msh-5 and rtel-1 mutants. (A) Incidence of unc-5(+) recombinants among progeny derived from eggs laid in the indicated time intervals phs; unc-5(+) recombinants were never detected in the 10- to 22-hour interval (corresponding to nuclei furthest in meiotic progression at the time of heat shock) in WT but were detected in this early interval in both msh-5 (*P = 0.007) and rtel-1 (**P = 0.003) mutant backgrounds. (B) Incidence of CO and NCO repair types (8).