Fundamental cell cycle kinases collaborate to ensure timely destruction of the synaptonemal complex during meiosis

Abstract

The synaptonemal complex (SC) is a proteinaceous macromolecular assembly that forms during meiotic prophase I and mediates adhesion of paired homologous chromosomes along their entire lengths. Although prompt disassembly of the SC during exit from prophase I is a landmark event of meiosis, the underlying mechanism regulating SC destruction has remained elusive. Here, we show that DDK (Dbf4-dependent Cdc7 kinase) is central to SC destruction. Upon exit from prophase I, Dbf4, the regulatory subunit of DDK, directly associates with and is phosphorylated by the Polo-like kinase Cdc5. In parallel, upregulated CDK1 activity also targets Dbf4. An enhanced Dbf4-Cdc5 interaction pronounced phosphorylation of Dbf4 and accelerated SC destruction, while reduced/abolished Dbf4 phosphorylation hampered destruction of SC proteins. SC destruction relieved meiotic inhibition of the ubiquitous recombinase Rad51, suggesting that the mitotic recombination machinery is reactivated following prophase I exit to repair any persisting meiotic DNA double-strand breaks. Taken together, we propose that the concerted action of DDK, Polo-like kinase, and CDK1 promotes efficient SC destruction at the end of prophase I to ensure faithful inheritance of the genome.

Keywords: Dbf4‐dependent Cdc7 kinase; Polo‐like kinase; homologous recombination; meiosis; synaptonemal complex.

Figure 1. An enhanced interaction between DDK and Cdc5 suppresses pachytene arrest

1. Schematic depicting the Cdc5 binding region of Dbf4. Residues in bold are essential for the interaction. +, wild‐type interaction; −, no interaction detected; ++, enhanced interaction.

2. Strains were induced to synchronously enter meiosis. At the indicated time points, cells were harvested for detection of proteins by immunoblotting (panels) and determination of cell cycle kinetics by DAPI staining of nuclei (graphs). Induction of Ndt80 and Cdc5 serves as a marker for pachytene exit. Total, total protein levels (Ponceau S staining). Mononucleate cells have not completed any nuclear divisions, binucleate cells have only completed the first nuclear division (anaphase I), and tri/tetranucleate cells have completed both nuclear divisions (anaphase I and II).

3. A fragment of Cdc5 containing the PBD was N‐terminally GST‐tagged and purified to near homogeneity, as determined by Coomassie staining (upper panel). Various Dbf4 peptides corresponding to sequences spanning the Cdc5 binding region were synthesized with a fluorescein tag (middle panel). Mutations are highlighted in gray. Measurements obtained from fluorescence polarization assays depicted in Appendix Fig S2B were used to calculate the dissociation constants (K _d) for each peptide‐PBD interaction (lower graphs). ND, not determined due to lack of detectable interaction (see Appendix Fig S2B).

4. Strains were induced to synchronously enter meiosis. Cells harvested at the indicated time points were used to examine the interaction between DDK and Cdc5 by immunoprecipitating Cdc7‐V5 using anti‐V5 antibody. WCE, whole‐cell extract. “−” and “+” indicate the exclusion and inclusion of antibody for IP, respectively, with the no antibody condition serving as a negative control.

Data information: At least 100 cells were scored per experiment. Data in (B) and (C) are represented as mean from two experiments and mean ± SEM from three experiments, respectively.Source data are available online for this figure.

Figure 2. The Cdc5‐Dbf4 fusion protein can suppress pachytene arrest

Either CDC5 or the CDC5‐dbf4‐R83E fusion construct was placed under the control of the DBF4 promoter and integrated at an ectopic locus (URA3) in the indicated strains. “–” denotes no ectopic insert.

1. Strains were induced to synchronously enter meiosis. Proteins were detected, and cell cycle kinetics was monitored as in Fig 1B.

2. Cells were incubated for 48 h on sporulation plates, and sporulation percentage was determined by light microscopy. White labels depict the native DBF4 locus, and gray labels depict the ectopic locus.

Data information: Data in (A) and (B) are represented as means from two and three experiments, respectively. Error bars in (B) indicate ± SEM. At least 100 cells were scored per experiment.Source data are available online for this figure.

Figure 3. DDK and Cdc5 interact to relieve Rad51 of its meiotic inhibition

1. A

   Strains were induced to synchronously enter meiosis. At the indicated time points, cells were harvested for analysis of meiotic chromosomes by pulsed‐field gel electrophoresis followed by Southern blotting with a probe recognizing chromosome II. Southern blots (panels) were quantified to determine the percentage of signal corresponding to broken chromosomes (graphs; see Materials and Methods).

2. B, C

   Strains were induced to synchronously enter meiosis. At 6 h, β‐estradiol was added to induce Cdc5 production (Cdc5 induction or Cdc5‐ind.). Cells were harvested at the indicated time points to (B) monitor meiotic chromosomes as in (A), or (C) detect proteins as in Fig 1B. “−” and “+” denote the absence or presence of an inducible CDC5 allele at the URA3 locus, respectively.

Data information: Data in (A) and (B) are represented as means from two experiments.Source data are available online for this figure.

Figure EV1. Rad51‐dependent DSB repair occurs before metaphase I

1. hop2Δ strains in the BR1919 background were transferred to sporulation media. At 22 h (hop2Δ rad17Δ) or 30 h (all other strains), cells were harvested and meiotic chromosomes were spread for immunofluorescence microscopy as in Fig 4B. Scale bar, 5 μm.

2. Quantification of the results in (A). The percentage of nuclei with metaphase spindle was determined for each strain; this represents cells that have exit pachytene and progressed into metaphase I. From nuclei with metaphase spindles, the percentage that are positive for DNA damage markers was determined; this represents cells that have progressed into metaphase I with unrepaired DSBs (e.g., the hop2Δ rad17Δ strain, in which the DNA damage checkpoint gene RAD17 has been deleted). NA, not applicable. At least 150 nuclei were scored for each strain.

Figure EV2. The Rad51‐dependent DSB repair facilitated by Dbf4‐Cdc5 does not efficiently produce interhomolog crossovers

1. Cells were incubated for 48 h on sporulation plates, and tetrads were dissected. After 3 days at 30°C, the number of colonies was counted and expressed as a percentage of spore viability. ND, not determined due to very low levels of sporulation; the spore viability of the dmc1Δ mutant was previously shown to be < 1% (Tsubouchi & Roeder, 2006).

2. A schematic of the HIS4‐LEU2 recombination hotspot that was utilized to monitor crossover formation. The site where DSBs are formed and the probe for Southern blotting are shown. Note the XhoI polymorphisms between parental chromosomes. rec, recombinant.

3. Strains were induced to synchronously enter meiosis. Cells were harvested at the indicated time points. Genomic DNA was extracted and digested with XhoI. Resultant DNA molecules were separated by gel electrophoresis and detected by Southern blotting using the probe shown in (B). rec, recombinant.

4. Quantification of the results in (C). The percentage of total DNA corresponding to recombinant DNA molecules was plotted. Data are represented as means from two experiments.

Source data are available online for this figure.

Figure 4. Cdc5‐dependent phosphorylation of Dbf4 drives SC disassembly

1. Strains were induced to synchronously enter meiosis. At 6 h, β‐estradiol was added to induce Cdc5 production. Proteins were detected as in Fig 1B. Images within the dotted boxes are expanded, and the signal quantified to illustrate the distribution of Dbf4 for that lane. The horizontal axis represents the percentage of signal, and the vertical axis corresponds to the source of that signal. The total area under the curve is set to be equal between strains. “−” and “+” denote the absence or presence of an inducible CDC5 allele at the URA3 locus, respectively.

2. ndt80Δ strains in the BR1919 background were transferred to sporulation media. At 20 h, β‐estradiol was added to induce Cdc5 production (confirmed in Fig EV3B). Cells were harvested, and meiotic chromosomes were spread for immunofluorescence microscopy at 2‐h intervals after addition of β‐estradiol. Representative images depicting the criteria for categorization of nuclei are shown (panels). Nuclei were categorized according to these criteria (graphs).

3. Spread meiotic chromosomes were prepared as in (B). Cells are from the same cultures as (B). A nucleus is shown with the polycomplex depicted by a white arrowhead (panels). Nuclei were categorized according to this criterion (graphs).

4. Strains in the BR1919 background containing NDT80‐6xHA were transferred to sporulation media. At 16 h, spread meiotic chromosomes were prepared as in (B). Representative images depicting the criteria for categorization of nuclei are shown (panels). Nuclei were categorized according to these criteria (graphs). *P < 0.05 (chi‐squared test), in comparison with the wild‐type ratio positive for both Zip1 and Ndt80.

Data information: Data in (B) and (C) are represented as means from two experiments. At least 100 nuclei were scored per experiment. At least 200 nuclei were scored for each strain in (D). All scale bars are 5 μm.Source data are available online for this figure.

Figure EV3. Production of Cdc5 leads to downregulation of DSB formation

1. A

   Strains were induced to synchronously enter meiosis. At 6 h, the dbf4‐E86V culture was split and either carrier (− Cdc5) or β‐estradiol (+ Cdc5) was added. The dbf4‐E86K culture only received carrier. Cells were harvested at the indicated time points, and proteins were detected as in Fig 1B.

2. B

   ndt80Δ strains in the BR1919 background were transferred to sporulation media. At 20 h, β‐estradiol was added to induce Cdc5 production. Cells were harvested at 2‐h intervals after addition of β‐estradiol, and proteins were detected as in Fig 1B. Cells are from the same culture as Fig 4B and C.

3. C

   Strains were induced to synchronously enter meiosis. Cells were harvested at the indicated time points, and proteins were detected as in Fig 1B. Total, total protein levels (Ponceau S staining).

4. D

   Cells were induced to synchronously enter meiosis. At 6 h, the culture was split and either carrier (− Cdc5 induction) or β‐estradiol (+ Cdc5 induction) was added. Cells were harvested at the indicated time points, and meiotic chromosomes were monitored as in Fig 3A. Data are represented as the results of an individual experiment (see Fig 5B for the duplicate experiment).

5. E, F

   Cells were induced to synchronously enter meiosis. At 3.5 h, the culture was split and either carrier (− Cdc5 induction) or β‐estradiol (+ Cdc5 induction) was added. Cells were harvested at the indicated time points to detect proteins as in Fig 1B (E) or monitor meiotic chromosomes as in Fig 3A (F). Cells are from the same culture. Data are represented as mean ± SEM from three experiments (*P < 0.05, paired t‐test).

Source data are available online for this figure.

Figure 5. Cdc5 kinase activity is required for destruction of SC components, unshackling of Rad51, and phosphorylation of Dbf4

1. A, B

   Strains were induced to synchronously enter meiosis. At 6 h, the culture was split and either carrier (Cdc5 induction −) or β‐estradiol (Cdc5 induction +) was added. Cells were harvested at the indicated time points to detect proteins as in Fig 1B (A) or monitor meiotic chromosomes as in Fig 3A (B). Cells are from the same cultures.

2. C, D

   Strains were induced to synchronously enter meiosis. At 6 h, β‐estradiol was added to induce production of Cdc5 or Cdc5‐kd (kinase dead, Cdc5‐N209A). Cells were harvested at the indicated time points to detect proteins as in Fig 1B (C) or monitor meiotic chromosomes as in Fig 3A (D). Cells from the cdc5‐kd set of strains are from the same cultures. “−” denotes the absence of an inducible CDC5 or cdc5‐kd allele at the URA3 locus.

3. E

   Strains were induced to synchronously enter meiosis. At 6 h, β‐estradiol was added to induce production of Cdc5. Cells were harvested at 10 h and resolved by SDS–PAGE in gels containing the indicated amounts of Phos‐tag reagent. Cells are from the same cultures as Fig 4A. “−” denotes the absence of an inducible CDC5 allele at the URA3 locus.

Data information: Data in (B) are represented as the results of an individual experiment (see Fig D for the duplicate experiment). Data in (D) are represented as means from two experiments.Source data are available online for this figure.

Figure 6. CDK1 kinase activity and DDK are required for efficient SC destruction

1. A, B

   Strains were induced to synchronously enter meiosis. At 4 h, cultures were split and either carrier or 1NM‐PP1 (an ATP analog that specifically inhibits Cdc28‐as1) was added (denoted “−” and “+” under 1NM‐PP1, respectively). Cells were harvested at the indicated time points, and proteins were detected as in Fig 1B.

2. C

   Strains were induced to synchronously enter meiosis. At 4 h, the culture was split and either carrier or 1NM‐PP1 was added. At 6 h, β‐estradiol was added to all cultures at the indicated concentrations to induce production of Cdc5. Cells were harvested at the indicated time points, and proteins were detected as in Fig 1B.

3. D, E

   Strains were induced to synchronously enter meiosis. In (D), β‐estradiol was added at 6 h to induce production of Cdc5. Cells were harvested at the indicated time points, and proteins were detected as in Fig 1B. The bob1 mutation was included as it bypasses the essential requirement for DDK in DNA replication.

4. F

   Strains were induced to synchronously enter meiosis. Cultures were split at 6 h, and either carrier (− rapamycin) or rapamycin (+ rapamycin) was added. β‐estradiol was added to all cultures at 8 h to induce production of Cdc5. Cells were harvested at the indicated time points, and proteins were detected as in Fig 1B. These strains were constructed in the anchor‐away background, where rapamycin triggers the nuclear deportation of FRB‐tagged proteins, which in this case corresponds to Cdc7 and Dbf4.

Source data are available online for this figure.

Figure EV4. The requirement for CDK1 in SC destruction is conditional

1. A, B

   Strains were induced to synchronously enter meiosis. At 4 h, cultures were split and either carrier or 1NM‐PP1 (an ATP analog that specifically inhibits Cdc28‐as1), was added (denoted “−” and “+” under 1NM‐PP1, respectively). Cells were harvested at the indicated time points, and proteins were detected as in Fig 1B.

Source data are available online for this figure.

Figure EV5. Conditional depletion of DDK before pachytene exit results in defective SC morphology

1. A

   Single colonies from the indicated strains were streaked on rich media with or without rapamycin. Since DBF4 and CDC7 are both essential genes, the observed growth defect in the presence of rapamycin indicates conditional inactivation of DDK.

2. B

   The ndt80Δ DBF4‐FRB strain was induced to synchronously enter meiosis. At 6 h, either carrier (control) or rapamycin (rapamycin) was added. Cells were harvested 2 and 4 h after drug treatment, and chromosomes were spread as in Fig 4B. To quantify these results, nuclei were scored for the presence of fully established SC, as judged by Zip staining, and the presence of Red1 staining (for representative images, see control nuclei). Nuclei in white boxes are expanded to the right. White arrowheads depict polycomplexes, which were larger than usual because of prolonged cell cycle arrest. At least 100 nuclei were scored in the presence and absence of rapamycin at each time point. Data are represented as means from two experiments. Scale bar, 5 μm.

3. C, D

   Strains were induced to synchronously enter meiosis. At 6 h, β‐estradiol was added to induce production of Cdc5. At the indicated time points, cells were harvested for detection of proteins as in Fig 1B (C). At the 7 and 8 h time points, cells were also harvested for immunoprecipitation with an anti‐Dbf4 antibody (D). 5× 4A denotes that fivefold more dbf4‐4A sample was loaded than DBF4. “−” and “+” indicate the exclusion and inclusion of antibody for IP, respectively, with the no antibody condition serving as a negative control. WCE, whole‐cell extract. Sol., soluble fraction.

4. E, F

   Strains were induced to synchronously enter meiosis. At 6 h, β‐estradiol was added to induce production of Cdc5. Cells were harvested at the indicated time points, and proteins were detected as in Fig 1B.

Source data are available online for this figure.

Figure 7. Phosphorylation of Dbf4 is integral to the timely destruction of SC proteins

1. A, B

   Strains were induced to synchronously enter meiosis. At 6 h, β‐estradiol was added to induce production of Cdc5. Cells were harvested at the indicated time points, and proteins were detected as in Fig 1B. dbf4‐4A encodes four Ser/Thr to Ala mutations (S318A, S319A, S374A, T375A).

2. C

   Schematic model of how the CDK‐DDK‐Polo axis facilitates the transition from meiotic prophase I to metaphase I. dep., dependent; pro, prophase I; meta, metaphase I; ana, anaphase I; HR, homologous recombination.

Source data are available online for this figure.