Meiosis employs two rounds of the cell division to produce haploid gametes (sperms or eggs), which are essential for sexual reproduction. During meiosis, programmed genetic recombination generates genetic diversity in organisms and ensures faithful chromosome segregation. Recombination events are initiated by meiosis-specific programmed double-strand break (DSB) formation regulated by the topoisomerase-like protein Spo11 (Keeney, 2001). DSBs undergo an additional process to expose 3′ single stranded DNA (ssDNA), which is mediated by the Exo1/Dna2 and Mre11/Rad50/Xrs2 complex (Cannavo and Cejka, 2014; Garcia et al., 2011). DSB ends with ∼500 nucleotide (nt) 3′ ssDNA tails preferentially interact with the homolog partner template rather than with the sister chromatid; these reactions are mediated by the RecA homolog proteins Dmc1 and Rad51 (Hong et al., 2013a; Lao et al., 2013). Dmc1 is a meiosis-specific recombinase found in most eukaryotes, including yeast, mice, and humans. Rad51 functions in homology searching and homolog pairing of DNAs during mitosis, but plays an auxiliary role in homology searching associated with Dmc1 during meiosis (Hong et al., 2013a). The dmc1Δ mutant in budding yeast shows an abnormal phenotype during recombination that reflects the essential role of this protein in meiotic recombination. A Dmc1-deficient strain exhibited defective DSB repair progression and deficient synaptonemal complex formation (Shinohara et al., 1997; Schwacha and Kleckner, 1997). It has been suggested that Dmc1 promotes DNA strand exchange to form synapsis exclusively between homologs, which is a process unique to meiotic recombination. In addition, DSBs accumulate in dmc1Δ mutants because of failed strand invasion, which triggers a checkpoint during meiotic prophase I. It has been reported that the Hop2-Mnd1 and Mei5-Sae3 complexes are dominant auxiliary factors for Dmc1 function and stimulate Dmc1-mediated ssDNA strand invasion during meiotic recombination (Chan et al., 2014; Ferrari et al., 2009). The Hop2-Mnd1 heterodimeric complex constructs a synaptic complex, which is a combination of 3′ ssDNA and homologous double-strand DNA in the D-loop formation stage (Chi et al., 2007; Kang et al., 2015; Pezza et al., 2007). Previous studies showed that in Saccharomyces cerevisiae, hop2Δ or sae3Δ mutant exhibits prophase arrest and DSB accumulation (Henry et al., 2006; Leu et al., 1998; Tsubouchi and Roeder, 2002). The Mei5-Sae3 heterodimeric complex is specifically expressed in meiotic cells (Hayase et al., 2004; Tsubouchi and Roeder, 2004). mei5Δ or sae3Δ mutant exhibits inefficient spore viability, prophase arrest, and defective recombinant products (Hayase et al., 2004; Tsubouchi and Roeder, 2004). Biochemical studies have shown that the Mei5-Sae3 complex promotes nucleation of Dmc1 onto ssDNA (Tsubouchi and Roeder, 2004). Therefore, the Hop2-Mnd1 and Mei5-Sae3 complexes may be essential for homolog bias mediated by Dmc1-dependent recombination in early prophase I.

Mek1 kinase is expressed specifically during meiosis and is activated by the induction of DSBs (Niu et al., 2007). Mek1 kinase activity inhibits inter-sister DSB repair during meiosis by directly affecting the sister chromatid axes. The pattern of meiotic recombination by Dmc1 and Rad51 shows that Mek1 kinase involves in Rad51-mediated recombination (Hong et al., 2013; Lao et al., 2013). Related studies revealed that Rad51’ strand exchange activity is inhibited by tightly controlled mechanisms that prevent the complex formation between Rad51 and Rad54 during meiotic recombination (Busygina et al., 2008). Thus, meiosis-specific Hed1 binds to Rad51, blocking the interaction with Rad54 and decreasing the binding affinity of Rad54 for Rad51 by phosphorylating Rad54 through Mek1 (Busygina et al., 2008; Niu et al., 2009). The choice of a partner for recombination during meiosis is highly modulated by interhomolog repair pathways after DSB resection to expose the 3′ end. Partner choice is modulated by Red1, Hop2, and Mek1 kinase activity (Hong et al., 2013a; Kim et al., 2010; Schwacha and Kleckner, 1997). Inactivation of Mek1 kinase promotes DSB repair to progress efficiently even in the absence of Dmc1; however, loss of homolog bias results in the repair of most DSBs via sister bias. In the absence of Mek1 kinase activity, cohesin channels the recombination to use the sister chromatid as a template, which is appropriate for mitotic DSB repair (mitotic mode) (Hong et al., 2013a). Thus, the sister chromatid is preferentially used as a partner template. This meiosis-specific pathway promotes homolog bias during the early stages post DSB, and additional pathways support that the bias is maintained during the interhomolog single-end invasion (IH-SEI) to interhomolog double-Holliday junctions (IH-dHJ) transition of interhomolog crossover (IH-CO) formation (Kim et al., 2010). The Hop2-Mnd1 complexes stimulate Dmc1-mediated strand exchange (Petukhova et al., 2005). Additionally, the Mei5-Sae3 complex co-localizes with Dmc1 and is required for Dmc1 function in vivo (Hayase et al., 2004; Tsubouchi and Roeder, 2004).

In this study, we investigated the roles of Hop2 and Sae3 during recombination in the presence or absence of Mek1 kinase activity, and found that both Hop2 and Sae3 promote homolog bias through a Dmc1-dependent pathway.

Yeast strains

Saccharomyces cerevisiae strains used in this study included SK1 derivatives isogenic with homozygous ho::hisG, leu2::hisG, ura3Δ (Pst1-Sma1). All genotypes and strain details are described in Supplementary Table 1.

Culture media and time course

The culture media and meiotic time course were essentially performed as previously described (Kim et al., 2010). Cells were patched to YPG plates (1% yeast extract, 2% peptone, 3% glycerol, and 2% bactoagar) for 24 h at 30°C. To select single colonies, cells from the YPG plate were streaked onto YPD plates (1% yeast extract, 2% peptone, 2% glucose, and 2% bactoagar) and grown at 30°C for 2 days. A single diploid colony resulting from this streaking was inoculated into 2 ml liquid YPD medium (1% yeast extract, 2% peptone, 2% glucose) and incubated at 30°C for 24 h. For synchronous meiosis, YPD cultures were inoculated in SPS medium (1% potassium acetate, 1% bactopeptone, 0.5% yeast extract, 0.17% yeast nitrogen without amino acids, 0.5% ammonium sulfate, 0.05 M potassium biphthalate, and 2 drops/L antifoam [Sigma, USA], pH 5.5) at a 1:500 dilution and cultured for 18 h. Meiosis was initiated in SPM medium (0.2% potassium acetate, 0.02% raffi-nose, and 2 drops/L antifoam). Meiotic cells were harvested and resuspended in 50 mM Tris-HCl and 50 mM EDTA. Cross-linking of cells was performed with psoralen under UV light for 10 min.

Physical analysis of meiotic recombination

Cultured cells at each time point were treated with Zymolyase (100T, US Biological, USA). Spheroplasted cells were subjected to guanidine-phenol extraction. DNA was precipitated with ethanol, and then resuspended in 50 mM Tris-HCl and 1 mM EDTA (Hong et al., 2013b; Kim et al., 2010). DNA concentration was measured using a Picogreen assay kit (Invitrogen, USA). For one-dimensional gel analysis, 2 μg genomic DNA was treated with XhoI (Enzynomics, Korea) for 3 h. Next, the DNA samples were loaded into a 0.6% Seakem LE agarose gel in TBE buffer at ∼2 V/cM for 24 h. For two-dimensional gel analysis, DNA digested in the same manner was then loaded into a 0.4% Seakem Gold agarose gel without ethidium bromide in TBE buffer at ∼1 V/cM for 21 h. After electrophoresis, the gel was stained for 30 min with 0.5 μg/ml ethidium bromide. Bands of interest were cut and arrayed on 2D gel trays. The gel was loaded in a two-dimensional manner. SeaKem LE agarose (0.8%) containing 0.5 μg/ml ethidium bromide was poured around the cut 1D gel array at 4°C. Gel electrophoresis was performed at ∼6 V/cM for 6 h at 4°C. For crossover and non-crossover gel analysis, 2 μg of genomic DNA was treated with both XhoI and NgoMIV followed by 1D gel analysis. Enzyme digestion and 1D gel loading conditions were the same as those described above for 1D and 2D gel loading. Southern hybridization was conducted using ³²P-dCTP-labeled radioactive nucleotides reacted with a random primer labeling mixture (Agilent Technologies, USA). Radioactive signals were observed using a Bio-Rad phosphoimager and quantified using Quantity One (Bio-Rad, USA).

Meiotic division curves

Cells from SPM cultures were harvested and fixed in 40% ethanol containing 0.1 M sorbitol. To count the cells undergoing meiosis I and II, DAPI stock solution was added to each cell fraction. Nuclei stained with DAPI (approximately 200 cells per each time point) were counted under a fluorescence microscope as previously described (Kim et al., 2010).

System for physical analysis of meiotic recombination

The progression of meiotic recombination was monitored by one-dimensional (1D) or two-dimensional (2D) agarose gel electrophoresis (Fig. 1; Hunter and Kleckner, 2001; Kim et al., 2010; Oh et al., 2007). Specifically, programmed DSB is initiated at the HIS4LEU2 locus only once in this position, and contains restriction polymorphisms to distinguish between the “Maternal” and “Parental” alleles as well as between meiotic recombination products (Fig. 1A). To obtain genomic DNA, the cells cultured in sporulation media were harvested at specific time points. The phenol-guanidine preparation method (Hong et al., 2013b; 2015; Kim et al., 2010; Koszul et al., 2008; Lee et al., 2015a) was used to prepare genomic DNA by Psoralen and UV cross-linking procedures, which involved treating each cell sample with psoralen and UV light exposure to fix the DNA interstrands. DNA was digested with the XhoI restriction enzyme and then subjected to 1D gel and 2D gel electrophoresis followed by Southern hybridization analysis to detect recombination intermediates and products (Figs. 1B and 1C). Meiotic recombination eventually produces IH-COs or interhomolog noncrossover (IH-NCO) without exchanging flanking genes (Fig. 1B; B?rner et al., 2004; Hong et al., 2013; Kim et al., 2010). After digestion with both XhoI and NgoMIV, IH-COs and IH-NCOs from the HIS4LEU2 locus were found to be 4.6 kb and 4.3 kb, respectively (Figs. 1A and 1B). IH and IS joint molecules (SEIs and dHJs) at the HIS4LEU2 locus showed different molecular weights and shapes, enabling differentiation between the SEI and dHJ stages by 2D gel electrophoresis (Figs. 1C and 1D; Lee et al., 2015b; Hong et al., 2013a; Hunter and Kleckner, 2001; Kim et al., 2010).

Meiotic DSB repair is defective in the absence of Hop2 and Sae3

The absence of Hop2 and Sae3 eliminates homologous chromosome synapsis and causes defects in DSB repair during meiotic recombination (Petukhova et al., 2003; Neale and Keerey, 2006). In a previous in vitro study, Hop2 showed distinct activity; the Hop2-Mnd1 complex stimulated Dmc1 and Rad51 recombinase activity, and purified Hop2 promoted strand invasion (Pezza et al., 2014). The Mei5-Sae3 complex interacts with Dmc1 to promote assembly of 3′ ssDNA on one side of a DSB (Gerton and Hawley, 2005). The physical analysis DNA in meiotic recombinant structures at the HIS4LEU2 locus has been used to examine the functions of Hop2 and Sae3 during meiotic recombination and their relationships with Mek1 kinase activity to understand the partner choice pathway. DSB formation in hop2Δ and sae3Δ mutants was analyzed by 1D gel electrophoresis (Fig. 2A). We further introduced the mek1as allele that is inactivated Mek1 kinase activity in the presence of 1-NA-PP1. All hop2Δ mek1as(?IN) and sae3Δ mek1as(?IN) cells showed defective DSB turnover, and DSB levels accumulated up to ∼36% compared to the control at 10 h, and hyper-resected DSBs were also observed (Fig. 2). These results suggest that Hop2 and Sae3 are essential for promoting the DSB-to-SEI transition during meiosis. However, in the presence of a chemical inhibitor (Mek1 kinase inactivation), DSBs were efficiently processed, but there was a large number of hyper-resected DSB. IH-CO levels were not effectively increased compared to wild-type levels. Additionally, this result implies that Mek1 kinase inactivation is associated with the progression of DSB repair, but IH-CO levels were not detectable (Fig. 2). Based on these results, we suggest that Hop2 and Sae3 are necessary for DSB repair during meiotic recombination.

Hop2 and Sae3 are involved in meiotic progression after DSB formation

DSBs were analyzed in the dmc1Δ mek1as, hop2Δ mek1as, and sae3Δ mek1as strains at the HIS4LEU2 locus where programmed DSBs are produced at one site (Figs. 1 and 3). As essential mediators, Exo1 and Mre11 together with Dna2 execute meiotic DSB end resection, specifically in the absence of Dmc1 (Garcia et al., 2011; Hong et al., 2013a; Lukaszewicz et al., 2015; Zakharyevich et al., 2010). dmc1Δ displays DSB accumulation and hyper-resection (Fig. 3A), whereas in dmc1Δ mek1as(+IN) strains, significantly reduced DSBs were observed in 2D gel analysis. Since Dmc1 plays a role in meiotic DSB repair, Rad51 critically functions in mitotic DSB repair in the absence of Mek1 kinase activity (Hong et al., 2013a). When Mek1 kinase was active in hop2Δ mek1as(-IN) and sae3Δ mek1as(-IN), DSB resection patterns resulted in hyper-resection of the DSB ends, leading to the production of extensive single-strand DNA tails at the 3′ end over time, as observed by 2D gel electrophoresis (Fig. 3). In the presence of Mek1 kinase activity, DSB levels remained elevated above the background signals detected in the wild-type strain because of a deficiency in DSB repair, causing DSB accumulation to higher stead-state levels. In contrast to mek1as(+IN), where Mek1 kinase was inactive, hop2Δ mek1as(?IN) and sae3Δ mek1as(?IN) showed early DSB hyper-resection, reaching maximum levels approximately 8?10 h into meiosis; DSBs were processed after 8 h (Fig 3B). Thus, Mek1 kinase inhibits DSB repair in the absence of Hop2 and Sae3, but when Mek1 kinase is inactivated, the cells progress efficiently into a DSB repair state via a pathway other than IH-CO progression (Figs. 2 and 4).

Hop2 and Sae3 are not required for joint molecule formation in the absence of Mek1 kinase activity

Hop2 plays a role in the DSB-to-SEI transition through Dmc1-mediated strand invasion. Hop2 is a meiosis-specific protein that localizes to the chromosomes to prevent joining between nonhomologous partners and further promote joining with a homologous partner (Pezza et al., 2007). Moreover, Hop2 functions as a heterodimer complex with Mnd1 to recognize partner template DNA and to mediate its repair during meiosis, particularly by homologous paring (Chan et al., 2014; Leu et al., 1998; Tsubouchi and Roeder, 2002). Sae3 is also an essential accessory factor that forms hetero-complex with Mei5 to assist Dmc1 to promote the meiotic DSB repair process (Hayase et al., 2004; Tsubouchi and Roeder, 2004). We investigated SEI and dHJ formation in wild-type, dmc1Δ mek1as, hop2Δ mek1as, and sae3Δ mek1as strains that were inactivated in the presence of I-NA-PPI inhibitor (Fig. 4). The wild-type cells showed 1.4% of IH-dHJ and 0.25% of IS-dHJ, and the ratio of IH:IS-dHJ was approximately 5:1. In the dmc1Δ mek1as(-IN) strain, both IH-dHJ and IS-dHJ were not detected in cells, whereas in dmc1Δ mek1as(+IN), the IH-dHJ levels peaked at 0.3% but IS-dHJ remained undetected (Fig. 4A and 4B). In the hop2Δ mek1as(?IN) strain, IH-dHJ levels were approximately 0.1%, but IS-dHJs were undetectable in cells; whereas hop2Δ mek1as (+IN) showed ∼0.1% IH-dHJ and ∼ 1% IS-dHJ, with a ratio of IH-dHJ to IS-dHJ approximately 1:9 (Fig. 4C and 4D; Supplementary Table 2). In the sae3Δ mek1as(?IN) strain, cells exhibited 0.1% IH-dHJ and 0.25% IS-dHJ, whereas sae3Δ mek1as(+IN) cells displayed close to 0% IH-dHJ and 0.75% IS-dHJ levels (Figs. 4C and 4D). Thus, Mek1 kinase inactivation triggered DSB repair and JM formation by converting the recombinational mode from IH bias to IS bias. Further, in mek1as(+IN) cells, SEIs appeared to form inter-sister arcs of SEI going to IS-dHJ (Fig. 4; Kim et al., 2010). Taken together, these results indicate that when Mek1 kinase was inactivated, cells underwent DSB repair through the inter-sister crossover (IS-CO) pathway. Thus, Mek1 kinase regulates recombination in the functional stages of Hop2 and Sae3 to convert from the mitotic mode into the meiotic mode.

Both Hop2 and Sae3 are required for the production of interhomolog recombination products

Homologous recombination leads to CO or NCO, the outcomes of meiotic DSB repair, in the flanking region of paired chromosomes (Allers et al., 2001; Mancera et al., 2008; Martini et al., 2006;). CO and NCO products can be distinguished at the HIS4LEU2 locus after digestion with XhoI and NgoMIV. In wild-type cells, the maximum levels of CO and NCO were 3.8% and 3.2%, respectively. However, in the hop2Δ mek1as and sae3Δ mek1as strains, regardless of whether 1-NA-PP1 inhibitor was present, the CO and NCO levels were less than 0.3% (Figs. 5A and 5B). Because hop2Δ mek1as(+IN) and sae3Δ mek1as (+IN) exhibit DSB hyper-resection and accumulation (Fig. 3B) without ongoing repair, low levels of COs and NCOs are expected. In contrast, DSBs in the hop2Δ mek1as(+IN) and sae3Δ mek1as(+IN) strains were repaired as in the wild-type strain (Fig. 3B) and the IS-dHJ level was highly increased compared to levels in cells without a 1-NA-PP1 inhibitor. Thus, since 1D gel analysis can only detect IH-COs or IH-NCOs, DSBs processed into IS-dHJ were resolved to IS-COs or IS-NCOs (Fig. 5C). This explains why DSB repair enters the mitotic mode, which uses sister templates rather than homologous templates, as in meiotic mode. Hence, Hop2 and Sae3 play roles in the fate of meiotic DSBs and determine whether programmed DSBs are processed by homolog bias or sister bias by Mek1 kinase. When Mek1 kinase activity is inhibited, the meiotic DSB repair procedure is directed towards the sister bias mechanism that use sister chromatids as templates (Tracy et al., 2010). In this case, DSBs are processed in the mitotic mode and are finally resolved with IS-NCO or IS-CO. However, more precise methods are required to distinguish these products. If Mek1 kinase plays the same role as in wild-type cells, the default option for programmed DSB repair is homologous bias, which uses homologous chromatids for recombination. When cells undergo defects in the SEI-to-dHJ transition, some of the SEIs may be converted into IH-NCO products, while SEI is stably converted into an IH-CO product when maintenance is sustained. Both Hop2 and Sae3 function during early prophase I before the SEI step as accessory factors of Dmc1, and therefore programmed DSBs are hyper-resected and accumulate because when subsequent pathways are blocked, such as in the absence of Hop2 or Sae3 (Fig. 5C).

In most organisms, recombinational interactions precisely occur between homologs to promote the efficient pairing of chromosomes and synapsis formation during meiosis. Dmc1 is a key factor in meiotic recombination and is required for homolog bias in the intrinsic pathway in meiosis. The budding yeast proteins Hop2 and Sae3 are assembled on DNA to promote heteroduplex DNA formation in a manner that affects Dmc1 strand exchange activity during meiotic recombination. Dmc1 and its accessory factors, Hop2 and Sae3, ensure that the first DSB ends are properly assembled onto the partner template to form a D-loop and that stably form SEI, which gives rise to the dHJ. These results suggest that during the DSB repair pathway, an early decision between the interhomolog and inter-sister pathway occurs, with most inter-sister CO arising from sister bias, and interhomolog CO events progressing to form an IH-dHJ. Further, IH pathway is switched to IS pathway when Mek1 kinase becomes inactive (Hong et al., 2013b). Thus, these results indicate that Hop2 and Sae3 require Mek1 kinase activity, which also plays a role in the post-DSB stage to promote Dmc1-mediated meiotic recombination.