The conserved ATPase, PCH-2/TRIP13, is required during both the spindle checkpoint and meiotic prophase. However, its specific role in regulating meiotic homolog pairing, synapsis and recombination has been enigmatic. Here, we report that this enzyme is required to proofread meiotic homolog interactions. We generated a mutant version of PCH-2 in C . elegans that binds ATP but cannot hydrolyze it: pch-2 E253Q . In vitro , this mutant can bind a known substrate but is unable to remodel it. This mutation results in some non-homologous synapsis and impaired crossover assurance. Surprisingly, worms with a null mutation in PCH-2’s adapter protein, CMT-1, the ortholog of p31 comet , localize PCH-2 to meiotic chromosomes, exhibit non-homologous synapsis and lose crossover assurance. The similarity in phenotypes between cmt-1 and pch-2 E253Q mutants suggest that PCH-2 can bind its meiotic substrates in the absence of CMT-1, in contrast to its role during the spindle checkpoint, but requires its adapter to hydrolyze ATP and remodel them.

Summary The conserved factor Shugoshin is dispensable in C. elegans for the two-step loss of sister chromatid cohesion that directs the proper segregation of meiotic chromosomes. We show that the C. elegans ortholog of Shugoshin, SGO-1, is required for checkpoint activity in meiotic prophase. This role in checkpoint function is similar to that of conserved proteins that structure meiotic chromosome axes. Indeed, null sgo-1 mutants exhibit additional phenotypes similar to that of a partial loss of function allele of the axis component, HTP-3: premature synaptonemal complex disassembly, the activation of alternate DNA repair pathways and an inability to recruit a conserved effector of the DNA damage pathway, HUS-1. SGO-1 localizes to pre-meiotic nuclei when HTP-3 is present but not yet loaded onto chromosome axes and genetically interacts with a central component of the cohesin complex, SMC-3, suggesting that it contributes to meiotic chromosome metabolism early in meiosis by regulating cohesin. We propose that SGO-1 acts during pre-meiotic replication to ensure fully functional meiotic chromosome architecture, rendering these chromosomes competent for checkpoint activity and normal progression of meiotic recombination. Given that most research on Shugoshin has focused on its regulation of sister chromatid cohesion during chromosome segregation, this novel role may be conserved but previously uncharacterized in other organisms. Further, our findings expand the repertoire of Shugoshin’s functions beyond coordinating regulatory activities at the centromere.

21 22The conserved factor Shugoshin is dispensable in C. elegans for the two-step loss of sister 23 chromatid cohesion that directs the proper segregation of meiotic chromosomes. We show that 24 the C. elegans ortholog of Shugoshin, SGO-1, is required for checkpoint activity in meiotic 25prophase. This role in checkpoint function is similar to that of the meiotic chromosomal protein, 26 HTP-3. Null sgo-1 mutants exhibit additional phenotypes similar to that of a partial loss of 27 function allele of HTP-3: premature synaptonemal complex disassembly, the activation of 28 alternate DNA repair pathways and an inability to recruit a conserved effector of the DNA 29 damage pathway, HUS-1. SGO-1 localizes to pre-meiotic nuclei, when HTP-3 is present but not 30 yet loaded onto chromosome axes, suggesting an early role in regulating meiotic chromosome 31 metabolism. We propose that SGO-1 acts during pre-meiotic replication to ensure fully 32 functional meiotic chromosome architecture, rendering these chromosomes competent for 33 checkpoint activity and normal progression of meiotic recombination. Given that most research 34

18 19 The conserved ATPase, PCH-2/TRIP13, is required during both the spindle checkpoint and 20 meiotic prophase. However, it's specific role in regulating meiotic homolog pairing, synapsis and 21 recombination has been enigmatic. Here, we report that this enzyme is required to proofread 22 meiotic homolog interactions. We generated a mutant version of PCH-2 in C. elegans that binds 23 ATP but cannot hydrolyze it: pch-2 E253Q . In vitro, this mutant binds its substrates but is unable to 24 remodel them. This mutation results in non-homologous synapsis and loss of crossover 25 assurance. Surprisingly, worms with a null mutation in PCH-2's adapter protein, CMT-1, the 26 ortholog of p31 comet , localize PCH-2 to meiotic chromosomes, exhibit non-homologous synapsis 27 and lose crossover assurance. The similarity in phenotypes between cmt-1 and pch-2 E253Q 28 mutants indicate that PCH-2 can bind its meiotic substrates in the absence of in 29 contrast to its role during the spindle checkpoint, but requires its adapter to hydrolyze ATP and 30 remodel them. 31Introduction 32 33 Sexual reproduction relies on meiosis, the specialized cell division that generates haploid 34 gametes, such as sperm and eggs, from diploid progenitors so that fertilization restores diploidy. 35To ensure that gametes inherit the correct number of chromosomes, meiotic chromosome 36 segregation is exquisitely choreographed: Homologous chromosomes segregate in meiosis I 37 and sister chromatids segregate in meiosis II. Having an incorrect number of chromosomes, 38 also called aneuploidy, is associated with infertility, miscarriages and birth defects, underscoring 39 the importance of understanding this process to human health. 40 41 Events in meiotic prophase ensure proper chromosome segregation. During prophase, 42 homologous chromosomes undergo progressively intimate interactions that culminate in 43 synapsis and crossover recombination (reviewed in [1]). After homologs pair, a macromolecular 44 complex, called the synaptonemal complex (SC), is assembled between them in a process 45 called synapsis. Synapsis is a prerequisite for crossover recombination to generate the 46 linkages, or chiasmata, between homologous chromosomes that direct meiotic chromosome 47 segregation. Defects in any of these events can result in chromosome missegregation during 48 the meiotic divisions and gametes, and therefore embryos, with an incorrect number of 49 chromosomes. 50 51The conserved AAA-ATPase PCH-2 (Pch2 in budding yeast, PCH2 in Arabidopsis and TRIP13 52 in mice) is crucial to coordinate these events in meiotic prophase. In vitro and cytological 53 experiments indicate that it does this by using the energy of ATP hydrolysis to remodel meiotic 54HORMADs [2][3][4][5][6], chromosomal proteins that are essential for pairing, synapsis, recombination 55 and checkpoint function [7][8][9][10][11][12][13][14][15][16][17][18]. HORMADs are a protein family defined by the ability of a 56 domain, the HORMA domain, to adopt multiple conformations that specify pr...

During meiotic prophase, the essential events of homolog pairing, synapsis, and recombination are coordinated with meiotic progression to promote fidelity and prevent aneuploidy. The conserved AAA+ ATPase PCH-2 coordinates these events to guarantee crossover assurance and accurate chromosome segregation. How PCH-2 accomplishes this coordination is poorly understood. Here, we provide evidence that PCH-2 decelerates pairing, synapsis and recombination in C. elegans by remodeling meiotic HORMADs. We propose that PCH-2 converts the closed versions of these proteins, which drive these meiotic prophase events, to unbuckled conformations, destabilizing interhomolog interactions and delaying meiotic progression. Further, we find that PCH-2 distributes this regulation among three essential meiotic HORMADs in C. elegans: PCH-2 acts through HTP-3 to regulate pairing and synapsis, HIM-3 to promote crossover assurance, and HTP-1 to control meiotic progression. In addition to identifying a molecular mechanism for how PCH-2 regulates interhomolog interactions, our results provide a possible explanation for the expansion of the meiotic HORMAD family as a conserved evolutionary feature of meiosis. Taken together, our work demonstrates that PCH-2’s remodeling of meiotic HORMADs has functional consequences for the rate and fidelity of homolog pairing, synapsis, recombination and meiotic progression, ensuring accurate meiotic chromosome segregation.

During meiotic prophase, the essential events of pairing, synapsis, and recombination are coordinated with meiotic progression to promote fidelity and prevent aneuploidy. The conserved AAA+ ATPase PCH-2 controls a variety of chromosome behaviors, including coordinating pairing, synapsis and recombination between homologous chromosomes to guarantee crossover assurance and accurate chromosome segregation. However, how PCH-2 accomplishes this coordination is poorly understood. Here, we use a combination of genetic, cytological, and biochemical analyses to show that PCH-2 regulates pairing, synapsis and recombination in C. elegans by remodeling meiotic HORMADs from a closed conformation to an unlocked conformation. Further, we find PCH-2 coordinates these events by distributing this regulation among the three essential meiotic HORMADs in C. elegans: PCH-2 acts through HTP-3 to regulate pairing and synapsis, HIM-3 to promote crossover assurance, and HTP-1 to control meiotic progression. In addition to identifying a molecular mechanism for how PCH-2 regulates interhomolog interactions, our results provide a possible explanation for the expansion of this family as a conserved evolutionary feature of meiosis. Taken together, our work demonstrates that PCH-2s well-characterized role in remodeling mitotic HORMADs applies to meiotic HORMADs and this remodeling coordinates homolog pairing, synapsis, recombination and meiotic progression to ensure accurate meiotic chromosome segregation.