Meiosis is the specialized form of cell division by which most sexually reproducing organisms generate haploid gametes. In a diploid organism, each meiotic cell begins prophase I with four copies of the genome – two homologous chromosomes (homologs) and identical copies of each homolog called sister chromatids. As mutations incurred in the gamete genome will be passed on to the resultant progeny, it is crucial that genome integrity be maintained during meiosis. Despite this risk, a highly conserved feature of the meiotic program is induction of DNA double strand breaks (DSBs) by the topoisomerase-like protein Spo11 (Keeney et al., 1997; Bergerat et al., 1997). A limited subset of DSBs must engage the homologous chromosome as a recombination partner and be repaired as a crossover event to forge a physical connection between homologs that facilitates accurate chromosome segregation at the meiosis I division. DSBs are incurred in excess of the number of eventual crossovers and other pathways must therefore be utilized to repair residual DSBs. How meiotic cells regulate repair pathway engagement to both accurately and efficiently resolve DSBs is a critical question in the field of genome integrity.
In many organisms, the majority of meiotic DSBs are repaired through interhomolog noncrossover recombination mechanisms (Hunter, 2015). Multiple models are proposed for how meiotic noncrossover repair occurs. Evidence in Drosophila suggests that both interhomolog noncrossovers and crossovers may be generated by differential processing of similar joint molecule intermediates (Crown et al., 2014). Work in budding yeast, mammals, and Arabidopsis indicates that the majority of interhomolog noncrossovers are generated via synthesis-dependent strand annealing (SDSA) with the homolog (Hunter, 2015). In SDSA, one or both resected end(s) of the DSB invades a repair template, new sequence is synthesized, the strand dissociates from its repair template, and finally utilizes the synthesized sequence to anneal to the other resected end of the DSB.
Meiotic DSBs may also be repaired by recombination with the sister chromatid (Goldfarb and Lichten, 2010; Toraason et al., 2021c; Almanzar et al., 2021; Schwacha and Kleckner, 1997). In budding yeast, DSB repair by intersister recombination is disfavored so as to promote recombination with the homologous chromosome (Goldfarb and Lichten, 2010; Schwacha and Kleckner, 1997; Humphryes and Hochwagen, 2014; Kim et al., 2010; Schwacha and Kleckner, 1994). In metazoan meiosis, however, the engagement of intersister repair has proven challenging to detect and quantify. While recombination between polymorphic homologs may be readily studied via sequence conversions in final repair products, the identical sequences of sister chromatids preclude the detection of intersister recombination by sequencing-based approaches. Recently, two methods have been developed in the nematode Caenorhabditis elegans to enable direct detection of homolog-independent meiotic recombination (Toraason et al., 2021c; Almanzar et al., 2021). Toraason et al., 2021c constructed an intersister/intrachromatid repair (ICR) assay that exploits nonallelic recombination at a known locus in the genome to identify homolog-independent repair events in resultant progeny (Figure 1-figure supplement 1 A). Almanzar et al., 2021 designed an EdU labeling assay to cytologically identify sister chromatid exchanges (SCEs) in compacted chromosomes at diakinesis. Together, these studies demonstrated that: (1) homolog-independent meiotic recombination occurs in C. elegans; (2) the sister chromatid and/or same DNA molecule is the exclusive recombination repair template in late prophase I; and, (3) intersister crossovers are rare and represent a minority of homolog-independent recombination products (Toraason et al., 2021c; Almanzar et al., 2021).
While meiotic cells primarily utilize homologous recombination to resolve DSBs, error prone repair pathways are also available in meiosis to repair DSBs at the risk of introducing de novo mutations (Gartner and Engebrecht, 2022). These error prone mechanisms are repressed in wild type contexts to promote recombination repair but are activated in mutants that disrupt recombination (Macaisne et al., 2018; Yin and Smolikove, 2013; Kamp et al., 2020; Lemmens et al., 2013). Non-homologous end joining (NHEJ), which facilitates the ligation of blunt DNA ends by the DNA ligase IV homolog LIG-4, is active in the C. elegans germline (Macaisne et al., 2018; Yin and Smolikove, 2013). Recent studies have revealed that microhomology-mediated end-joining facilitated by the DNA polymerase θ homolog POLQ-1 (theta-mediated end-joining, TMEJ) is the primary pathway by which small mutations are incurred in C. elegans germ cells and somatic cells (Kamp et al., 2020; van Schendel et al., 2015). Since neither NHEJ nor TMEJ are required for successful meiosis (Kamp et al., 2020; Lemmens et al., 2013; Volkova et al., 2020; Colaiácovo et al., 2003), recombination is likely sufficient for meiotic DSB repair and gamete viability under normal conditions.
The structural maintenance of chromosomes 5/6 complex and tumor suppressor BRCA1 (SMC-5/6 and BRC-1 respectively in C. elegans) are highly conserved and regulate meiotic DSB repair in C. elegans (Kamp et al., 2020; Bickel et al., 2010; Hong et al., 2016; Li et al., 2018; Janisiw et al., 2018; Odiba et al., 2024). The SMC-5/6 complex is vital for preservation of meiotic genome integrity, as C. elegans mutants for smc-5 exhibit a transgenerational sterility phenotype (Bickel et al., 2010). Although null mutations of smc-5, smc-6, and brc-1 revealed that they are not required for development nor reproduction in C. elegans (Bickel et al., 2010; Li et al., 2018; Janisiw et al., 2018; Adamo et al., 2008), both SMC-5/6 and BRC-1 are required for efficient DSB repair, as smc-5 and brc-1 null mutants both display meiotic chromosome fragmentation at diakinesis indicative of unrepaired DSBs (Bickel et al., 2010). In C. elegans, BRC-1 has also been shown to repress error prone DSB repair via NHEJ and TMEJ (Kamp et al., 2020; Li et al., 2020), and acts in parallel with the tumor suppressor 53BP1/HSR-9 (Hariri et al., 2023). Further, SMC-5/6 and BRC-1 may promote genome integrity in part by facilitating efficient recombination, as smc-5 and brc-1 mutants exhibit persistent DSBs marked by the recombinase RAD-51 (Kamp et al., 2020; Bickel et al., 2010; Adamo et al., 2008; Boulton et al., 2004), suggesting that early recombination steps are disrupted in these mutants. BRC-1 further prevents recombination between heterologous templates to promote accurate recombination repair (León-Ortiz et al., 2018). Despite these apparent DNA repair defects, interhomolog crossover formation is largely unaffected by smc-5 and brc-1 mutations (Bickel et al., 2010; Li et al., 2018; Janisiw et al., 2018; Adamo et al., 2008). Taken together, these data have contributed to the model that that SMC-5/6 and BRC-1 are required for intersister repair in C. elegans.
SMC-5/6 and BRC-1 genetically interact to regulate germline DSB repair. The incidence of unrepaired DSBs observed in both smc-5 and brc-1 single mutants are not additive in the double smc-5;brc-1 mutant context, which suggests that SMC-5/6 and BRC-1 may share some DSB repair functions (Bickel et al., 2010). Other experiments, however, indicate opposing functions for SMC-5/6 and BRC-1, as both the mitotic DNA replication defects in smc-5 mutants and the synthetic lethality of smc-5;him-6 (BLM helicase) double mutants are suppressed by brc-1 mutation (Hong et al., 2016; Wolters et al., 2014). Crucially, the specific steps of recombination regulated by SMC-5/6 and BRC-1 that intersect to influence DNA repair outcomes remain unknown.
To determine the DSB repair functions of SMC-5 and BRC-1 that regulate DNA repair outcomes during C. elegans meiosis, we employed a multipronged approach utilizing genetic assays, cytology, sequence analysis of recombinant loci, and functional DSB repair assays in smc-5 and brc-1 mutants. We find that SMC-5 and BRC-1 function to repress meiotic intersister crossover recombination, and that BRC-1 specifically regulates homolog-independent noncrossover intermediate processing. Through these experiments, we also find that BRC-1 prevents mutagenic DSB repair at the mid-pachytene stage of meiotic prophase I. By assessing germ cell capacity to resolve exogenous DSBs, we demonstrate that meiotic nuclei become more dependent on BRC-1 for DSB repair in late stages of meiotic prophase I. Finally, we reveal that smc-5 mutant DSB repair defects are suppressed by loss of brc-1, which impedes gamete viability in part by repressing error prone repair pathways and promoting recombination. Taken together, our study defines specific functions and interactions of BRC-1 and SMC-5 that regulate meiotic DSB repair outcomes across meiotic prophase I.