Multiple mechanisms limit meiotic crossovers: TOP3α and two BLM homologs antagonize crossovers in parallel to FANCM Mathilde Séguéla-Arnauda,b,1, Wayne Crismania,b,1, Cécile Larchevêquea,b, Julien Mazela,b, Nicole Frogera,b, Sandrine Choinarda,b, Afef Lemhemdia,b, Nicolas Macaisnea,b, Jelle Van Leenec,d, Kris Gevaerte,f, Geert De Jaegerc,d, Liudmilla Chelyshevaa,b, and Raphael Merciera,b,2 aInstitut Jean-Pierre Bourgin, UMR1318, équipes de recherche labellisées CNRS 3559, National Institute for Agricultural Research, Saclay Plant Sciences, 78000 Versailles, France; bInstitut Jean-Pierre Bourgin, UMR 1318, équipes de recherche labellisées CNRS 3559, AgroParisTech, Saclay Plant Sciences, 78000 Versailles, France; cDepartment of Plant Systems Biology, Flanders Institute for Biotechnology, B-9052 Ghent, Belgium; dDepartment of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium; eDepartment of Medical Protein Research, Flanders Institute for Biotechnology, B-9000 Ghent, Belgium; and fDepartment of Biochemistry, Ghent University, B-9000 Ghent, Belgium Edited by R. Scott Hawley, Stowers Institute for Medical Research, Kansas City, MO, and approved February 23, 2015 (received for review December 4, 2014) Meiotic crossovers (COs) have two important roles, shuffling genetic species: the Bloom syndrome (BLM) homolog, small growth information and ensuring proper chromosome segregation. Despite suppressor 1 (Sgs1) in Saccharomyces cerevisiae (3, 4); regulator their importance and a large excess of precursors (i.e., DNA double- of telomere elongation helicase1 (RTEL-1) in Caenorhabditis strand breaks, DSBs), the number of COs is tightly regulated, typi- elegans (5), and the Fanconi anemia of complementation group cally one to three per chromosome pair. The mechanisms ensuring M (FANCM) helicase in Arabidopsis and Schizosaccharomyces that most DSBs are repaired as non-COs and the evolutionary forces pombe (6, 7). These helicases are thought to displace the in- imposing this constraint are poorly understood. Here we identified vading strand, allowing its annealing with the other 3′ overhang α α — Arabi- Topoisomerase3 (TOP3 ) and the RECQ4 helicases the end of the DSB, leading to NCO formation in a process called dopsis slow growth suppressor 1 (Sgs1)/Bloom syndrome protein “synthesis-dependent strand annealing” (SDSA). Nevertheless, (BLM) homologs—as major barriers to meiotic CO formation. First, the characterization of a specific TOP3α mutant allele revealed that, even when CO formation is increased threefold by the disruption in addition to its role in DNA repair, this topoisomerase antagonizes of AtFANCM, DSBs still greatly outnumber COs (6), suggesting CO formation. Further, we found that RECQ4A and RECQ4B consti- the existence of additional anti-CO pathways. tute the strongest meiotic anti-CO activity identified to date, their Results and Discussion concomitant depletion leading to a sixfold increase in CO frequency. In both top3α and recq4ab mutants, DSB number is unaffected, and The meiotic anti-CO activity of FANCM was identified through extra COs arise from a normally minor pathway. Finally, both TOP3α a genetic screen because its mutation restores bivalent formation and RECQ4A/B act independently of the previously identified and fertility of zmm mutants (6). To identify additional meiotic anti-CO Fanconi anemia of complementation group M (FANCM) anti-CO factors, we extended this screen and isolated one helicase. This finding shows that several parallel pathways actively suppressor of human enhancer of invasion-10 (hei10), hei10(s)61, limit CO formation and suggests that the RECQA/B and FANCM helicases prevent COs by processing different substrates. Despite Significance a ninefold increase in CO frequency, chromosome segregation was unaffected. This finding supports the idea that CO number is re- During meiosis, crossovers (COs) reshuffle homologous chromo- stricted not because of mechanical constraints but likely because somes, generating genetic diversity on which natural or human of the long-term costs of recombination. Furthermore, this work selection can act. However, CO numbers typically are very low, demonstrates how manipulating a few genes holds great promise raising questions about the evolutionary forces that impose this for increasing recombination frequency in plant-breeding programs. constraint and limiting the efficiency of breeding programs. Here, we identified anti-CO factors in Arabidopsis and showed that recombination | meiosis | crossover | Topoisomerase 3 | RECQ4 several mechanisms actively antagonize CO formation in parallel. Disrupting these anti-CO factors provokes a large increase in CO eiotic homologous recombination is initiated by the for- frequency without affecting meiotic progression. These results GENETICS Mmation of DNA double-strand breaks (DSBs). DSBs suggest that COs are restrained not because a high number are resected to form 3′ ssDNA overhangs which invade the would impair chromosome segregation but because excessive intact homologous chromosome, producing DNA joint molecules recombination could break favorable genetic combinations (JMs). These JMs can be differentially processed to produce built by past selection. These findings hold great promise for crossovers (COs) or non-COs (NCOs). In Arabidopsis thaliana, improving the efficiency of plant breeding programs. mammals, and budding yeast, two pathways of CO formation exist. The major pathway depends on the ZMM proteins (for Author contributions: M.S.-A., W.C., and R.M. designed research; M.S.-A., W.C., C.L., J.M., N.F., S.C., A.L., N.M., J.V.L., G.D.J., and L.C. performed research; K.G. contributed new Zip1-4, Msh4/5, and Mer3) in addition to MutL homolog 1 reagents/analytic tools; M.S.-A., W.C., K.G., and R.M. analyzed data; and M.S.-A., W.C., (MLH1) and MuL homolog 3 (MLH3) and produces interfering and R.M. wrote the paper. COs, so that one CO prevents the formation of another nearby Conflict of interest statement: A provisional patent based on the work has been filed by (1). The second, pathway, producing noninterfering COs, the National Institute for Agricultural Research. depends on structure-specific endonucleases including MUS81 This article is a PNAS Direct Submission. (1). These pro-CO pathways compete with anti-CO pathways, Freely available online through the PNAS open access option. resulting in a minor portion of DSBs becoming COs; for instance, 1M.S.-A. and W.C. contributed equally to this work. it is estimated that COs represent only 10% and 5% of DSBs in 2To whom correspondence should be addressed. Email: [email protected]. mouse and in Arabidopsis, respectively (2). Three helicases with This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. meiotic anti-CO activities have been identified in different 1073/pnas.1423107112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1423107112 PNAS | April 14, 2015 | vol. 112 | no. 15 | 4713–4718 Downloaded by guest on September 30, 2021 with increased fertility and bivalent number (Fig. 1A). The were described previously (8). The top3α-1 mutation leads to combination of genetic mapping, whole-genome sequencing, and lethality early in development; top3α-2, a hypomorphic mutant, is functional complementation (24 independent transformants) viable but shows stunted growth and complete sterility, the latter identified the recessive causal mutation in Topoisomerase3α being linked to aberrant bivalent-like structures followed by (TOP3α; At5g63920). In addition to hei10, this mutation was able massive fragmentation at meiosis, suggesting an accumulation of to restore fertility and bivalent formation of mutS homolog 5 unresolved JMs. The severe phenotypes associated with TOP3α (msh5) (Fig. 1A), showing that TOP3α prevents CO formation in disruption in Arabidopsis and various other species (9–12) zmm mutants. The hei10(s)61 mutation changes Arg640 into a highlight its essential role in the resolution of mitotic and meiotic stop codon (top3α-R640X hereafter). Therefore, TOP3α-R640X DNA-repair intermediates. The phenotypes conferred by the has intact topoisomerase-primase (TOPRIM) and topoisomerase top3α-R640X mutation contrast with the previously described domains, but two predicted zinc-finger domains, which are con- alleles: the top3α-R640X plants did not show any somatic defect served in TOP3α in plants and animals but not in yeasts, are (Fig. S2), showed no fragmentation at meiosis (Fig. 1B), and were truncated (Fig. S1). In Arabidopsis, two mutant alleles of TOP3α fertile (Table S1). The increased number of bivalents observed in *** *** N.S. *** ** *** *** *** *** *** *** *** *** *** *** *** *** *** *** A *** *** *** N.S. *** *** *** *** N.S. *** N.S. 5 4 per meiosis per 3 2 Univalents / 1 Bivalents 0 (n=17) (n=92) (n=30) (n=35) (n=28) (n=27) (n=35) (n=23) (n=44) (n=48) (n=37) (n=36) (n=27) (n=36) (n=20) (n=36) (n=91) -R640X -2 (n=33) α -2 (n=48) α α msh4 -R640X msh5-2 hei10-2 -R640X shoc1-1 /top3 /top3 α Wild type (n=29) α hei10(S)61 top3 hei10-2 top3 msh4 recq4a-4 msh4 recq4b-2 -R640X -R640X fancm-1 -R640X α α α -R640X recq4a-4 -R640X spo11-1-3 α recq4a-4 recq4b-2 α msh5-2 top3 top3 top3 top3 top3 msh4recq4b-2 recq4a-4 shoc1-1 recq4a-4 recqb-2 recq4a-4 recq4b-2 fancm-1 recq4a-4 recqb-2 spo11-1-3 B top3 hei10-2 Wild type hei10 hei10 top3α-R640X top3α-R640X top3α-2 hei10 top3α-2 b b a b a a u a b u u a a a a a a u b b Metaphase I Anaphase I Anaphase 25 C D F 45 *** N.S. *** 40 *** 8 ) 20 *** 7 35 *** 30 6 15 5 *** 25 *** 4 10 N.S. *** 20 *** *** *** 3 15 2 ** 10 N.S. N.S. N.S. N.S. Gentic size (cM) 5 Genetic size (cM Genetic size 1 5 Genetic size (cM) 0 0 0 Fig. 1. Meiotic recombination is increased in the top3α- I2b I2a I2b I2a I1b I1c I5c I5d I2b I2a I5c I5d R640X and recq4a recq4b mutants. (A) Average number of hei10 hei10 Wild type top3α-R640X top3α-R640X/top3α-1 Wild type recq4a recq4b recq4a top3α-R640X recq4b bivalents per male meiocyte.