Introduction

Cohesin-mediated sister chromatid cohesion occurs during meiotic S-phase to prevent premature separation of sister chromatids. Following the loss of cohesins from chromosome arms, homologous chromosomes are segregated to opposite poles during meiosis I; after the degradation of cohesins at centromeres during meiosis II, sister chromatids are segregated to opposite poles [6]. Mitotic chromosome determinant 1 (Mcd1) is the α-kleisin subunit of the cohesin complex that is required for sister chromatid cohesion during mitosis. Saccharomyces cerevisiae Mcd1 was originally identified in genetic screens as a factor that is essential for chromosome structure and cell viability [3]. Temperature-sensitive Mcd1 (ts-Mcd1) mutants have defects in chromosome condensation, leading to precocious separation of sister chromatids [4]. Several lines of evidence suggest that Mcd1 forms discrete foci on meiotic pachytene chromosomes that connect sister chromatids [12]. By analogy, Saccharomyces cerevisiae Rec8 cohesin is the meiosis-specific α-kleisin subunit of the meiotic cohesin complex that mediates chromosome organization and meiotic recombination [1,13]. Rec8 is capable of synaptonemal complex (SC) assembly and double-strand break (DSB) repair [17,14], but not in mitotic cells [5]. This functional divergence raises the possibility that Mcd1 can partially substitute for Rec8 but also has unique roles in regulating the progression of meiotic intermediates during meiosis.

Homologous chromosomes are the preferred templates for DSB repair during meiosis in diploid yeast cells [9,10]. In contrast, during mitosis, sister chromatids are preferentially used as the template [11]. During this process, cohesin tightly binds to sister DNA molecules until the onset of anaphase following S-phase to ensure correct sister chromatid separation [18]. In both meiotic and mitotic cells, cohesion at chromatid axes is required for DSB repair [15,24]. The present study investigated the functional divergence of Mcd1 by examining whether the mitotic cohesin subunit Mcd1 regulates meiotic recombination in budding yeast. We ectopically expressed Mcd1 protein by replacing its promoter with that of Cup1, which is strongly activated by copper. As a result, meiotic division was delayed and sister chromatid separation proceeded more slowly. Moreover, we have investigated meiosis-specific recombination molecules, DSB, single-end invasion (SEI), double-Holliday junction (dHJ), and crossover (CO), and analyzed the kinetics of recombination intermediates during meiosis [7,8,13]. Using a DNA physical analysis technique, we confirmed that Mcd1 regulates the recombination progress in the formation and resolution of meiotic joint molecules (JMs) into final recombination products. Together, these results provide new insights into the roles of Mcd1 at mitosis-to-meiosis transition and the progression of meiotic recombination even in the presence of Rec8.

Materials and Methods

Meiotic Time Course

Diploid Saccharomyces cerevisiae cells were patched onto yeast extract-peptone-glycerol medium (1% (w/v) yeast extract, 2% (w/v) bacto-peptone, 3% (w/v) glycerol, and 2% (w/v) bacto agar) for 15 h. Patched cells were streaked on agar yeast extract-peptone-dextrose (YPD) plates (1% (w/v) yeast extract, 2% (w/v) bacto peptone, 2% (w/v) dextrose, and 2% (w/v) bacto agar) and grown for an additional 48 h. A single colony was cultured in liquid YPD medium for 24 h. To synchronize cells at the G1 phase, cultures were diluted 1:500 (v/v) in 200 ml of supplemented presporulation medium (SPS; 0.5% (w/v) yeast extract, 1% (w/v) bacto peptone, 0.67% (w/v) yeast nitrogen base without amino acids, 1% (w/v) potassium acetate, and 0.05 M potassium biphtalate, adjusted to pH 5.5 with 10 N KOH) at 30℃ for 18 h, and cells were washed with 200 ml of sporulation medium (SPM) (1% (w/v) potassium acetate, 0.025% (w/v) raffinose, and 0.015% (v/v) antifoam) and then resuspended in 200 ml of SPM to initiate meiosis. Cells were harvested at the desired time points and centrifuged at 3,000 rpm for 2 min. For Mcd1 expression in the pCUP1-Mcd1 strain, 50 µM CuSO4 (from 20 mM CuSO4 in distilled water) was added to SPM cultures derived from the same SPS culture for 2.5 h.

DNA Physical Analysis

To stabilize JMs, cells were mixed with 0.1 mg/ml trioxsalen (Sigma-Aldrich, St. Louis, CA, USA) and exposed to 360 nm ultraviolet radiation for 15 min, and then resuspended in Tris-EDTA buffer (50 mM Tris-HCl and 50 mM EDTA, pH 8.0) and centrifuged at 3,000 rpm for 2 min. Genomic DNA was extracted as previously described [7,8,9,13,21,22,23]. For 1D gel analysis, genomic DNA was digested with 80 U of XhoI (Enzynomics, Daejeon, Korea) at 37℃ for 3 h and precipitated with >99% ethanol. Digested DNA was dissolved in DNA loading buffer and resolved on a 0.6% Seakem agarose gel (Takara Bio Inc., Otsu, Japan) in TBE (89 mM Tris-Borate and 2 mM EDTA, pH 8.3) at 2 V/cm for 24 h. The gel was stained with TBE containing 0.5 µg/ml ethidium bromide (EtBr) for 30 min. For 2D gel analysis, 3 µg of genomic DNA was digested with 80 U of XhoI, precipitated, and dissolved as described above. The digested DNA was resolved on a 0.4% Seakem agarose gel in TBE for 21 h at 25 V. The gel was stained with TBE containing 0.5 µg/ml EtBr. DNA bands were excised, placed in a 2D gel tray, and separated on a 0.8% agarose gel in TBE buffer containing 0.5 µg/ml EtBr at 120 V for 6 h. The gel was treated with 0.25 M HCl for 20 min and 0.4 M NaOH for 30 min, and then transferred to a Zeta-probe GT membrane (Bio-Rad, Hercules, CA, USA) for 18 h at room temperature. Probes were radiolabeled with 32P-dCTP using a random priming kit (Agilent Technologies, Santa Clara, CA, USA). Hybridized DNA species were detected with a phosphoimager and quantified using Quantity One software (Bio-Rad).

Meiotic Division

Meiotic divisions were visualized in cells stained with 4’,6-diamidino-2-phenylindole by fluorescence microscopy using an Olympus BX53 epifluorescence microscope (Olympus, Tokyo, Japan). Cells were harvested at each time point and immediately fixed in 0.1 M sorbitol and 40% ethanol and then stored at −20℃. A total of 200 cells were counted at each time point [19].

Western Blotting Assay

Yeast cell protein lysates were prepared as previously described [16]. Meiotic cells were harvested and centrifuged at 5,000 rpm for 1 min, and the pellet was resuspended in 100 µl of distilled-water; 100 µl of 0.6 M NaOH was then added to a final concentration of 0.3M NaOH. Samples were incubated at room temperature for 5 min, and then centrifuged at 5,000 rpm for 1 min. Each pellet was resuspended in Laemmli buffer and boiled at 95℃ for 5 min. Lysates were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Mcd1 was detected using an anti-HA antibody (sc-7392; Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:2,000.

Analysis of Sister Chromatid Cohesion

Meiotic cells containing the Tet operator/repressor-green fluorescent protein (TetO/TetR-GFP) array on chromosome II were harvested at each time point and fixed in 0.1 M sorbitol and 40% ethanol and stored at −20℃ [13]. Fixed cells were centrifuged at 3,000 rpm for 2 min and resuspended in Tris-EDTA buffer (10 mM-Tris and 1 mM EDTA, pH 8.0). Sister chromatid separation was monitored by TetO/TetR-GFP fluorescence and images were acquired using IS Capture Application software (Tucsen, Fuzhou, China) on an epifluorescence microscope.

Yeast Strains

All strains used in this study were on the SK1 background. The HIS4LEU2 locus and corresponding restriction sites [7-9,13,21-23]. are shown in Fig. 2. Strains were homozygous for MATa/MATα, ho::hisG, leu2, and ura3. The genotypes of the strains used for physical analysis of DNA and cohesion analysis were nuc1::hygroB/”, HIS4::LEU2-(BamHI+ori)/his4-x::LEU2-(NgoMIV+ori)-URA3, pCUP1-3HA-KanMX-Mcd1 (KKY1061), and lys2::TetOx240:URA3, leu2::LEU2 tetR-GFP, pCUP1-3HA-KanMX-Mcd1 (KKY1539), respectively.

Results and Discussion

Mcd1 Suppresses Normal Meiotic Division and Sister Chromatid Cohesion

The Mcd1 protein is repressed during meiosis, but small amounts of Mcd1 protein are present as cells transition from mitosis to meiosis. To investigate the role of Mcd1 during meiosis, we constructed a heterogeneous pCUP1-Mcd1 diploid strain by mating the wild type (WT) and pCUP1-Mcd1 haploid strain. In the latter, the endogenous Mcd1 promoter was replaced by the CUP1 promoter, enabling the expression of Mcd1 protein with an N-terminal 3HA-tag in the presence of copper (Fig. 1) [26]. After synchronizing mitotic cells at the G1 phase with SPS medium, cells were transferred to SPM to initiate synchronous meiosis; 2.5 h later, 50 µM CuSO4 was added to the pCUP1-Mcd1 culture to induce the expression of Mcd1 protein. The expression of 3HA-Mcd1 protein was detected after the addition of copper to the culture and persisted until 10 h after sporulation (Fig. 1A).

Fig. 1.Effect of Mcd1 expression on meiotic division and association of sister chromatids. (A) Induction of Mcd1 protein in S. cerevisiae strain pCUP1-Mcd1 during meiosis. Mcd1 expression was detected using an antibody against HA. Mcd1 was overexpressed by adding CuSO4 at 2.5 h after the induction of meiosis. A black arrow indicates the time when CuSO4 was added. (B) Schematic illustration of the TetO/TetR array system. The centromeres are indicated in black circles. Representative images of TetO/TetR-GFP are shown at the bottom. (C) Quantitative analysis of meiotic division and sister chromatid segregation. Blue line, meiotic division; red line, two GFP foci.

To investigate whether Mcd1 functions as a cohesin subunit during meiosis, we examined chromosome segregation by sister chromatid cohesion analysis [1,13]. A previous study found that the WT and ts-Mcd1 mutant had ~100% normal chromosome segregation during meiosis, although the segregation was slower in the latter [14]. When sister chromatid separation was examined using the LacO/LacI-GFP system, a single GFP signal per nucleus was detected after DNA replication, and two signals appeared after meiotic division I; however, the rec8 deletion mutant exhibited defective segregation, with two GFP signals observed after DNA replication [13,14]. Moreover, the Mcd1 protein persisted through meiosis and was observed connecting sister chromatids [12]. These results suggest that a deficiency in the meiotic cohesin Rec8 leads to chromosome mis-segregation, and that its mitotic counterpart Mcd1 is required for sister chromatid cohesion along with Rec8 during meiosis.

To examine the role of Mcd1 during meiosis, we experimentally induced the expression of Mcd1 protein by adding CuSO4 to pCUP1-Mcd1 cultures. Sister chromatid cohesion was examined in a strain carrying 240 tandem arrays of TetO and TetR fused to GFP that binds TetO repeats at one locus of chromosome II in the absence or presence of Mcd1 (Fig. 1B). A single GFP focus indicates unreplicated or replicated but unseparated sister chromatids, whereas double GFP foci represent replicated, fully separated sister chromatids. Meiotic division was delayed about 2 h in pCUP1-Mcd1 (+CuSO4) (Fig. 1C, blue line) as compared with pCUP1-Mcd1 (−CuSO4) cells. After the completion of meiosis (24 h in Fig. 1C), nuclear divisions had occurred in 97% of pCUP1-Mcd1 (−CuSO4) and 79% of pCUP1-Mcd1 (+CuSO4) cells. Furthermore, the fraction of pCUP1-Mcd1 (+CuSO4) cells with double GFP foci was reduced, with maximum values of 52% vs. 68% in pCUP1-Mcd1 (−CuSO4) cells, suggesting that the delay in sister chromatid separation was dependent on meiotic progression in Mcd1-expressing cells (Fig. 1C, red line). That is, the progression through meiotic division was correlated with sister chromatid separation. Therefore, Mcd1 prevents both meiotic division and sister chromatid separation during meiosis. These results imply that Mcd1 and Rec8 may compete for binding to chromosomes, or that Mcd1 interferes with the binding of Rec8 to chromosomes by occupying binding sites while remaining in uncleaved form.

Mcd1 Promotes DSB Initiation But Prevents Crossover Formation in Meiosis

When Mcd1 protein expression was induced during meiosis, sister chromatid replication and separation were delayed by about 2 h and progressed more slowly (Fig. 1). To evaluate the function of Mcd1 in meiotic recombination, we performed 1D gel electrophoresis and analyzed the formation of DSBs and COs between parental (Mom and Dad) chromosomes at the HIS4LEU2 hotspot of chromosome III during meiosis (Figs. 2A and 2B).

Fig. 2.Physical analysis of meiotic recombination in pCUP1-Mcd1 cells. (A) Map of the HIS4LEU2 locus showing diagnostic restriction and probe sites. The XhoI restriction site is indicated. Mom and Dad signals were detected at 5.9 and 4.3 kb, respectively; DSB signals were detected at 3.3 and 3 kb of the HIS4LEU2 locus on chromosome III. Probe 4 was used for Southern blotting analysis. (B) Representative image of 1D gel. JMs, joint molecules; COs, crossovers; DSBs, double-strand breaks. (C) Representative image of 1D gel of pCUP1-Mcd1 cells with or without addition of CuSO4 during meiosis. Mcd1 protein expression was induced by adding 50 µM CuSO4 at 2.5 h after the initiation of meiosis. (D) Quantitative analysis of DSBs and COs in (C).

Cells were synchronized in the G1 phase prior to initiation of meiosis. Cells were then exposed to ultraviolet light to stabilize DNA recombinants by intrastrand crosslinking, and total genomic DNA samples were purified by guanidine-phenol extraction [7-9,13,21,22]. Genomic DNA was digested with XhoI, and DNA fragments were analyzed by 0.6% agarose gel electrophoresis followed by Southern blotting using Probe 4 (Fig. 2A). DSB signals from Mom and Dad chromosomes were detected at 3.3 and 3 kb, respectively, of the HIS4LEU2 locus of chromosome III (Fig. 2A). Mom (CO-1) and Dad (CO-2) recombinant signals were detected at 5.6 and 4.6 kb, respectively, by 1D gel analysis (Figs. 2A and 2B). In pCUP1-Mcd1 (−CuSO4) cells, DSBs began to appear 3.5 h after the induction of meiosis and persisted up to 24 h after sporulation (Figs. 2C and 2D). COs emerged 6 h after the induction of meiosis and were present until its completion (Figs. 2C and 2D). When CuSO4 was added to meiotic cultures of pCUP1-Mcd1 at 2.5 h, the initiation of DSB formation occurred about 1 h sooner, as DSB turnover was accelerated. However, the CO levels were reduced to about 12% when Mcd1 was expressed during meiosis, although COs appeared about 1 h sooner (Figs. 2C and 2D). The delay in meiotic progression may have decreased CO formation in pCUP1-Mcd1 (+CuSO4) cells (Fig. 1C) owing to the abundant expression of M cd1 following DSB formation. The small amounts of Mcd1 protein remaining at the early stages of meiosis and the requirement for Mcd1 in the local sister chromatid connection suggest that Mcd1 may participate in initial DSB formation by supporting sister chromatids until Rec8 protein is expressed at sufficient levels. Indeed, Mcd1 disappeared and Rec8 appeared 2 h after the induction of meiosis (data not shown), suggesting that Mcd1 supports DSB formation but negatively regulates meiotic progression in post-DSB stages.

Mcd1 Expression Delays the Formation of Single-End Invasions and Double-Holliday Junctions

JMs arising from meiotic recombination at the HIS4LEU2 hotspot can be distinguished and analyzed by 2D gel electrophoresis based on molecular weight and shape (Figs. 2A and 3A). JMs originate from DSBs, which differentiate into SEIs and subsequently become dHJs between homologous chromosomes or sister chromatids (interhomolog (IH) and intersister (IS) interactions, respectively) (Fig. 3A). We showed that Mcd1 expression during meiosis had no effect on DSB formation but reduced the CO frequency (Fig. 2). To determine whether Mcd1 influences the progression of homologous recombination after DSB formation, we induced Mcd1 protein expression during meiosis with copper and monitored JM formation by 2D gel electrophoresis. Mcd1 extended the lifespan of SEIs and dHJs (Figs. 3B and 3C), with the levels increasing by about 0.5% for both types of intermediates in pCUP1-Mcd1 (+CuSO4) cells as compared with pCUP1-Mcd1 (-CuSO4) cells (Fig. 3C). Rad51 is a key protein in the homologous recombination process, which functions in homolog recognition and strand exchange during meiosis. It has been reported that the ratio of IH:IS dHJ in WT cells is 5:1 and 1:7 in cells deficient in Rad51 [7,8]. However, the IH:IS dHJ ratio was 5:1 and was unaltered in the presence of Mcd1 (Figs. 3B and 3C); a high frequency of SEIs remained even 24 h after induction of meiosis. When the level of each type of meiotic intermediate and JM was normalized by setting the maximal value of each intermediate to 100%, three important observations were made concerning meiotic progression as a function of Mcd1 expression (Fig. 4A). First, Mcd1 normally functions in the initiation of DSB formation during meiosis. Second, in post-DSB stages, Mcd1 not only affects the formation of SEIs but also delays their resolution and, consequently, the turnover of IH-dHJs and IS-dHJs (Fig. 4). Finally, JMs (SEIs and dHJs) are not completely resolved to the final products (COs) until 24 h (Fig. 1D). In summary, all meiotic events related to Mcd1 expression, including maximal peak time of SEIs, IH-dHJ, and IS-dHJ formation, were delayed by about 1 h in pCUP1-Mcd1 (+CuSO4) cells; however, Mcd1 expression accelerated the maximal peak time of DSB formation from 6.8 to 6 h (Fig. 4B). Taken together, it could be possible that the delay on JM to CO transition causes an accumulation of SEIs, IH-dHJ, and IS-dHJ.

Fig. 3.Ectopic expression of Mcd1 delays the formation of SEIs and dHJs during meiosis. (A) Representative image of a 2D gel of WT cells showing dHJs and SEIs. (B) Representative images of 2D gel analysis of pCUP1-Mcd1 cells with or without addition of CuSO4 during meiosis. (C) Quantitative analysis of JMs (Joint molecules). Black line, single-end invasion (SEI); blue line, interhomolog double-Holliday junction (IH-dHJ); red line, intersister double-Holliday junction (IS-dHJ); broken black line, total double-Holliday junction (T-dHJ).

Fig. 4.Timing and kinetics of meiotic recombination in the presence or absence of Mcd1. (A) Normalization of DSB, SEI, and dHJ levels. All JMs were included in the calculation (the level at each time point with respect to the level at the maximum peak time point. Black line, +CuSO4 ; broken grey line, −CuSO4. (B) Maximum time of DNA species in the presence or absence of Mcd1.

For the accurate meiotic recombination, homologous chromosomes and sister chromatids should be held together until meiosis I and II, respectively. Rec8 cohesin in meiosis and Mcd1 cohesin in mitosis have the important role of meiotic progression by tightly holding chromosomes. When cohesin protein is eliminated in meiosis, the localization of Zip1 protein onto the chromosomes is impaired, and then the assembly of the synaptonemal complex becomes abrogated. It was previously reported that when Rec8 was substituted with Mcd1 during meiosis, Zip1 assembly was severely compromised, implying the failure of SC assembly [2] possibly due to delayed or abnormal Zip1 chromosomal localization. Zip1 is a meiosis-specific SC protein, which is essential for chromosome synapsis and cell cycle progression in budding yeast [25]. Checkpoint activation at leptoteneto-zygotene/pachytene may be related to this abnormality in meiotic recombination, which prevents sister chromatid segregation [20]. In this study, the ectopic expression of Mcd1 delayed meiotic division, sister chromatid separation, and JM formation under circumstances in which Rec8 protein is normally expressed. Consequently, the formation of meiotic intermediates was delayed and meiotic recombinants did not fully dissolve until the end of meiosis. Alternatively, other surveillance mechanisms may be responsible for the defects resulting from Mcd1 expression during meiosis [6]. In contrast to the delay in JM formation, the turnover of DSB formation was accelerated when Mcd1 was expressed during meiosis. This functional divergence between Mcd1 and Rec8 during vegetative growth and meiotic division requires further investigation.

Meiotic recombination events are triggered by DSB formation in leptotene of meiotic prophase I. Subsequently, recombination proceeds though zygotene until the formation of a nascent D-loop and SEIs/dHJs during pachytene, followed by the formation of recombinants before organized chromosomes are ultimately diffused (Fig. 5). The presence of Mcd1 during meiosis delays the DSB-JM transition; prophase I proceeds normally until the DSB formation stage, but recombination events are inefficient from the latter stage of leptotene. In conclusion, Mcd1 regulates meiotic division and sister chromatid separation by causing a delay in the formation and resolution of branched joint molecules.

Fig. 5.Progression of meiotic recombination in the presence of Mcd1. Schematic illustration of chromosome organization and DSB-to-CO formation during meiotic prophase I. During leptotene, DSBs are initiated and axes develop. Nascent D-loops form and stabilize, becoming SEIs during zygotene. Successive strand exchange at the DSB ends yields dHJs during pachytene. During the pachytene-todiffuse stage, dHJs are resolved into recombinant products. Mcd1 promotes DSB formation, but the progression of DSB repair is delayed in post-DSB stages when high levels of Mcd1 accumulate during meiosis.