Fundamental cell cycle kinases collaborate to ensure timely destruction of the synaptonemal complex during meiosis
2017; Springer Nature; Volume: 36; Issue: 17 Linguagem: Inglês
10.15252/embj.201695895
ISSN1460-2075
AutoresBilge Argunhan, Wing‐Kit Leung, Negar Afshar, Yaroslav Terentyev, Vijayalakshmi V. Subramanian, Yasuto Murayama, Andreas Hochwagen, Hiroshi Iwasaki, Tomomi Tsubouchi, Hideo Tsubouchi,
Tópico(s)Mitochondrial Function and Pathology
ResumoArticle10 July 2017free access Source DataTransparent process Fundamental cell cycle kinases collaborate to ensure timely destruction of the synaptonemal complex during meiosis Bilge Argunhan orcid.org/0000-0002-6023-7654 Genome Damage and Stability Centre, Life Sciences, University of Sussex, Brighton, East Sussex, UK Institute of Innovative Research, Tokyo Institute of Technology, Tokyo, Japan Search for more papers by this author Wing-Kit Leung Genome Damage and Stability Centre, Life Sciences, University of Sussex, Brighton, East Sussex, UK Search for more papers by this author Negar Afshar Genome Damage and Stability Centre, Life Sciences, University of Sussex, Brighton, East Sussex, UK Institute of Innovative Research, Tokyo Institute of Technology, Tokyo, Japan Search for more papers by this author Yaroslav Terentyev Genome Damage and Stability Centre, Life Sciences, University of Sussex, Brighton, East Sussex, UK Search for more papers by this author Vijayalakshmi V Subramanian orcid.org/0000-0003-4037-3832 Department of Biology, New York University, New York, NY, USA Search for more papers by this author Yasuto Murayama Institute of Innovative Research, Tokyo Institute of Technology, Tokyo, Japan Search for more papers by this author Andreas Hochwagen Department of Biology, New York University, New York, NY, USA Search for more papers by this author Hiroshi Iwasaki Institute of Innovative Research, Tokyo Institute of Technology, Tokyo, Japan Search for more papers by this author Tomomi Tsubouchi Corresponding Author [email protected] Genome Damage and Stability Centre, Life Sciences, University of Sussex, Brighton, East Sussex, UK National Institute for Basic Biology, Okazaki, Japan Search for more papers by this author Hideo Tsubouchi Corresponding Author [email protected] orcid.org/0000-0003-0814-8432 Genome Damage and Stability Centre, Life Sciences, University of Sussex, Brighton, East Sussex, UK National Institute for Basic Biology, Okazaki, Japan Search for more papers by this author Bilge Argunhan orcid.org/0000-0002-6023-7654 Genome Damage and Stability Centre, Life Sciences, University of Sussex, Brighton, East Sussex, UK Institute of Innovative Research, Tokyo Institute of Technology, Tokyo, Japan Search for more papers by this author Wing-Kit Leung Genome Damage and Stability Centre, Life Sciences, University of Sussex, Brighton, East Sussex, UK Search for more papers by this author Negar Afshar Genome Damage and Stability Centre, Life Sciences, University of Sussex, Brighton, East Sussex, UK Institute of Innovative Research, Tokyo Institute of Technology, Tokyo, Japan Search for more papers by this author Yaroslav Terentyev Genome Damage and Stability Centre, Life Sciences, University of Sussex, Brighton, East Sussex, UK Search for more papers by this author Vijayalakshmi V Subramanian orcid.org/0000-0003-4037-3832 Department of Biology, New York University, New York, NY, USA Search for more papers by this author Yasuto Murayama Institute of Innovative Research, Tokyo Institute of Technology, Tokyo, Japan Search for more papers by this author Andreas Hochwagen Department of Biology, New York University, New York, NY, USA Search for more papers by this author Hiroshi Iwasaki Institute of Innovative Research, Tokyo Institute of Technology, Tokyo, Japan Search for more papers by this author Tomomi Tsubouchi Corresponding Author [email protected] Genome Damage and Stability Centre, Life Sciences, University of Sussex, Brighton, East Sussex, UK National Institute for Basic Biology, Okazaki, Japan Search for more papers by this author Hideo Tsubouchi Corresponding Author [email protected] orcid.org/0000-0003-0814-8432 Genome Damage and Stability Centre, Life Sciences, University of Sussex, Brighton, East Sussex, UK National Institute for Basic Biology, Okazaki, Japan Search for more papers by this author Author Information Bilge Argunhan1,2, Wing-Kit Leung1, Negar Afshar1,2, Yaroslav Terentyev1, Vijayalakshmi V Subramanian3, Yasuto Murayama2, Andreas Hochwagen3, Hiroshi Iwasaki2, Tomomi Tsubouchi *,1,4 and Hideo Tsubouchi *,1,4 1Genome Damage and Stability Centre, Life Sciences, University of Sussex, Brighton, East Sussex, UK 2Institute of Innovative Research, Tokyo Institute of Technology, Tokyo, Japan 3Department of Biology, New York University, New York, NY, USA 4National Institute for Basic Biology, Okazaki, Japan *Corresponding author. Tel : +81 564 557693; E-mail: [email protected] *Corresponding author. Tel : +81 564 557695; E-mail: [email protected] EMBO J (2017)36:2488-2509https://doi.org/10.15252/embj.201695895 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Synopsis Synaptonemal complex (SC) disassembly during yeast meiosis is induced by Cdc5- and Cdc28-mediated activation of Dbf4-dependent kinase (DDK), and is crucial for correct meiotic chromosome segregation by reactivating Rad51-dependent homologous recombination repair of persisting meiotic DNA double-strand breaks after prophase. DDK regulates timely destruction of the SC. DDK-associated SC degradation is controlled through phosphorylation of its regulatory subunit Dbf4. The interaction between Dbf4 and Polo-like kinase Cdc5 promotes Dbf4 phosphorylation and SC destruction. Cdc5 and cyclin-dependent kinase Cdc28 collaborate to achieve robust Dbf4 phosphorylation at the prophase I-metaphase I transition. SC destruction activates Rad51-dependent homologous recombination. Introduction Meiosis is central to the continuity of life in sexually reproducing organisms through the production of gametes. In meiosis, a single round of DNA replication is followed by two successive rounds of nuclear division, leading to the reduction of genetic material by exactly half. The unique aspect of meiosis lies in meiosis I, where homologous chromosomes (homologs) separate; this is in sharp contrast to mitosis or meiosis II, where sister chromatids separate (Petronczki et al, 2003). Meiotic chromosomes undergo dynamic morphological changes as homologs align with one another (Cahoon & Hawley, 2016). Sister chromatids are organized around a proteinaceous axis, which is juxtaposed at close proximity along its entire length with the axis of the homolog. The incorporation of a proteinaceous transverse filament between axes leads to the formation of a meiosis-specific chromosomal structure called the synaptonemal complex (SC). By adhering homologous axes in such a way, the SC provides a structural platform to promote efficient formation of crossovers between homologs, a process that is catalyzed by the homologous recombinases Rad51 and Dmc1. The function and structure of the SC have been the subject of extensive research in budding yeast (Tsubouchi et al, 2016). SC components associated with chromosomal axes are highly relevant for repressing usage of Rad51, which is involved in homologous recombination (HR) during both mitosis and meiosis (Shinohara et al, 1992). Unlike Rad51, Dmc1 is only produced during meiosis, where it is thought to play a specialized role in promoting interhomolog interactions in meiotic HR (Bishop et al, 1992). Thus, preferential usage of Dmc1 serves to promote interhomolog HR (Schwacha & Kleckner, 1997; Lao et al, 2013). Red1 and Hop1, structural components of meiotic chromosome axes, and Mek1, a meiosis-specific protein kinase functioning with Red1 and Hop1, are essential for repressing HR in the absence of Dmc1 (Schwacha & Kleckner, 1997; Wan et al, 2004). The phosphorylation of Hop1, which is under the control of the recombination checkpoint (see below), is also critical for repressing Rad51 (Carballo et al, 2008). Given the central role of the SC in regulating meiotic HR, it is of particular importance to understand the regulation of SC dynamics. Prophase I is divided into substages based on SC morphology (Roeder, 1997). During early-prophase I, newly replicated homologs start to condense (leptotene) and pairing of homologs initiates SC formation (zygotene). SC formation is considered complete when all paired homologs are incorporated along their entire lengths into the SC structure in mid-prophase I (pachytene). The SC is then disassembled in late prophase I (diplotene), before entry into metaphase I. SC behavior during the passage from pachytene to diplotene (referred to as pachytene exit hereafter) warrants special attention as it encompasses the time when SC disassembly takes place. Timely SC disassembly is essential for proper segregation of homologs at anaphase I, as the SC would otherwise oppose the microtubule forces that are responsible for separating homologs (Cahoon & Hawley, 2016). Pachytene exit also coincides with the maturation of recombination intermediates into interhomolog crossovers (Sourirajan & Lichten, 2008). Consistently, the timing of pachytene exit is closely coordinated with the progression of HR by the recombination checkpoint, also known as the pachytene checkpoint (Hochwagen & Amon, 2006). The recombination checkpoint is highly related to the DNA damage checkpoint operating in mitotic cells, except that a major downstream target of the signaling cascade is Ndt80, a meiosis-specific transcriptional activator that governs the mid-to-late stages of meiosis and sporulation including pachytene exit (Xu et al, 1995; Chu et al, 1998). Budding yeast cells that progress past the recombination checkpoint make an irreversible commitment to meiosis and swiftly disassemble the SC as they enter metaphase I (Tsuchiya et al, 2014). Thus, pachytene exit represents a key event in the prophase I–metaphase I transition and commitment to the meiotic nuclear divisions. The mechanisms governing SC disassembly have just begun to emerge. One major factor is Cdc5 (homolog of PLK1 and the only Polo-like kinase in budding yeast), whose production is induced in an Ndt80-dependent manner as cells exit pachytene (Chu et al, 1998; Sourirajan & Lichten, 2008; Acosta et al, 2011; Okaz et al, 2012). Cdc5 has also been shown to play a central role in regulating the resolution of recombination intermediates in both mitosis and meiosis (Sourirajan & Lichten, 2008; Matos et al, 2011, 2013; Szakal & Branzei, 2013). Production of Cdc5 before pachytene exit triggers untimely disassembly of the SC and resolution of recombination intermediates (Sourirajan & Lichten, 2008), arguing that Cdc5 is a major regulator of these events. During the prophase I–metaphase I transition, Cdc5 was shown to interact with another fundamental cell cycle kinase complex called Dbf4-dependent Cdc7 kinase (DDK; Matos et al, 2008). DDK has drawn comparisons to cyclin-dependent kinases (CDKs) as Cdc7 comprises the catalytic subunit, whereas Dbf4 fulfills a crucial regulatory role within the complex (Matthews & Guarné, 2013). Although its major role in vegetative cells is in controlling the initiation of DNA replication, DDK also has meiosis-specific roles in DSB formation and chromosome segregation (Matos et al, 2008; Sasanuma et al, 2008; Wan et al, 2008; Murakami & Keeney, 2014). However, unlike Cdc5, which functions after pachytene exit (Okaz et al, 2012), DDK is believed to function primarily before pachytene exit. Here, we show that DDK is central to the control of SC destruction in budding yeast. Dbf4 serves as the regulator of SC destruction by directly associating with and being phosphorylated by Cdc5. In parallel, Dbf4 is also regulated through phosphorylation by Cdc28 (homolog of CDK1 and the only CDK in budding yeast). We propose that the concerted action of DDK, CDK1, and Polo ensures SC destruction, with Dbf4 serving as the hub of the signaling pathway at the prophase I–metaphase I boundary, leading to the timely removal of a major physical obstacle to chromosome segregation. Interestingly, this coordinated mechanism leads to the reactivation of Rad51, which promotes the repair of any persisting DSBs before chromosomes are separated during anaphase I. By facilitating removal of the SC and triggering Rad51-dependent DSB repair, we propose that fundamental cell cycle kinases collaborate at the prophase I–metaphase I transition to ensure faithful inheritance of the genome. Results DDK and Polo interact to regulate the meiotic cell cycle Meiotic HR is initiated by the topoisomerase-like protein Spo11, which continuously forms meiotic DNA double-strand breaks (DSBs) before pachytene exit (Keeney et al, 1997). Unlike mitotic HR, meiotic HR is intricately regulated so that homologous chromosomes are connected through crossovers. Defects in meiotic HR lead to an accumulation of recombination intermediates, such as DSBs, which slowdown or arrest the meiotic cell cycle. In order to obtain further insight into the mechanism coordinating meiotic HR with cell cycle progression, we conducted a genetic screen to identify genes whose overexpression bypassed the cell cycle arrest caused by defects in meiosis-specific recombination factors (see Materials and Methods for experimental details). This screen revealed that overexpression of DBF4 can suppress pachytene arrest in several recombination mutants (Appendix Fig S1A). To understand how a high dose of Dbf4 suppresses the cell cycle arrest phenotype, we set out to isolate DBF4 point mutants that phenocopy this suppression effect. Randomly mutagenized versions of DBF4 were cloned into a single-copy plasmid to produce a DBF4 mutant library. Clones that were able to suppress the cell cycle arrest phenotype were screened. A single clone, carrying a mutation that changes the Glu at the 86th position to Val, was isolated (dbf4-E86V hereafter; Appendix Fig S1B). This amino acid falls within residues 83–88 at Dbf4's N-terminus, which were previously shown to mediate the direct interaction between Dbf4 and the Polo-box domain (PBD) of Cdc5 (Fig 1A; Chen & Weinreich, 2010). Of the residues within this region, Arg83, Ile85, Gly87, and Ala88 are essential for the interaction, whereas Glu86 is not. Furthermore, the mutant polypeptide with Glu86 changed to Lys (dbf4-E86K hereafter) interacts more strongly with Cdc5 than the wild-type polypeptide (Chen & Weinreich, 2010). These observations raised the possibility that the mechanism responsible for suppression of meiotic arrest involves an interaction between Dbf4 and Cdc5. Thus, we employed two known DBF4 mutations: Arg83 to Glu (dbf4-R83E hereafter) and dbf4-E86K, which abolish and enhance the Dbf4-Cdc5 interaction, respectively (Fig 1A; Chen & Weinreich, 2010). dbf4-R83E did not suppress cell cycle arrest, whereas dbf4-E86K showed a similar level of suppression to dbf4-E86V (Appendix Fig S1C), leading to the robust upregulation of late-stage proteins associated with cell cycle progression (Ndt80 and Cdc5) in dmc1Δ, a recombination deficient mutant that undergoes pachytene arrest (Fig 1B). Figure 1. An enhanced interaction between DDK and Cdc5 suppresses pachytene arrest Schematic depicting the Cdc5 binding region of Dbf4. Residues in bold are essential for the interaction. +, wild-type interaction; −, no interaction detected; ++, enhanced interaction. 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). 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 (Kd) for each peptide-PBD interaction (lower graphs). ND, not determined due to lack of detectable interaction (see Appendix Fig S2B). 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. Source Data for Figure 1 [embj201695895-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint To test whether suppression of cell cycle arrest by dbf4-E86K/V requires Cdc5, we wanted to deplete Cdc5 during meiosis. Since CDC5 is an essential gene, deletion mutants are not viable, so a conditional mutant was generated instead by transplacement of the native CDC5 promoter with the CLB2 promoter (cdc5-md, meiotic depletion; Lee & Amon, 2003). CLB2 is expressed during vegetative growth but downregulated during meiosis. Under this condition, Cdc5 was barely detectable within prophase I and the dbf4-E86K mutation was no longer able to suppress the pachytene arrest of the dmc1Δ mutant (Appendix Fig S2A), confirming the requirement for Cdc5 in Dbf4-mediated suppression of cell cycle arrest. The identical suppression phenotype of dbf4-E86K and dbf4-E86V suggested that, like the E86K mutation (Chen & Weinreich, 2010), the E86V mutation enhances the interaction between Dbf4 and Cdc5. To directly test this possibility, the fluorescence polarization assay was employed. The interaction strength between the C-terminal half of Cdc5 containing the Polo-box domain (PBD) and polypeptides corresponding to residues 73–96 of Dbf4 (wild type and mutants) was determined (Fig 1C, see Appendix Supplementary Methods for experimental details). Consistent with previous work (Chen & Weinreich, 2010), the wild-type Dbf4 peptide interacted with Cdc5-PBD with a Kd of ~2 μM (Fig 1C and Appendix Fig S2B). The E86K and E86V peptides showed a stronger interaction than wild type, with Kd values of ~0.3 μM, while the R83E peptide showed little/no interaction (Appendix Fig S2B), suggesting that Dbf4-E86K/V proteins interact more strongly with Cdc5 than wild-type Dbf4. To validate these in vitro results and further correlate Dbf4-Cdc5 interaction strength with suppression of pachytene arrest, we performed co-immunoprecipitation (co-IP) experiments to examine Dbf4-Cdc5 complex formation in meiosis. Cdc7 was C-terminally tagged with 9× copies of the V5 epitope and introduced into a genetic background in which cells arrest uniformly at the end of metaphase I due to meiosis-specific depletion of the anaphase promoting complex/cyclosome (APC/C) activator Cdc20 (cdc20-md; Matos et al, 2008). Cdc7 was immunoprecipitated from DBF4, dbf4-R83E, and dbf4-E86K/V strains 5 h and 6.5 h into meiosis, when Cdc5 levels were low and high, respectively. Cdc7, Dbf4, and Cdc5 were then detected by immunoblotting. Importantly, comparable amounts of Dbf4 were seen to co-IP with Cdc7 in all four strains (Fig 1D), suggesting that the mutations examined here do not affect the interaction between Cdc7 and Dbf4. In sharp contrast, the amount of Cdc5 that co-IP'd with Dbf4-E86K/V was increased at both time points compared to wild-type Dbf4. Furthermore, very low levels of Cdc5 were seen to co-IP with Dbf4-R83E, even at 6.5 h, when intracellular Cdc5 levels were high. These in vitro and in vivo data indicate that the dbf4-E86K/V mutations enhance DDK-Cdc5 complex formation, whereas the dbf4-R83E mutation reduces DDK-Cdc5 complex formation (Fig 1C and D, Appendix Fig 2B). We also noted that the migration of both Cdc7 and Dbf4 was affected by the dbf4 mutations. Posttranslational modification of Dbf4 was subjected to further investigation (see below). We also created a condition where the Dbf4-Cdc5 interaction is forced by fusing the two genes in-frame and expressing this fusion construct within prophase I by employing the DBF4 promoter. The Cdc5-Dbf4 fusion protein did not interfere with wild-type meiosis, as judged by spore viability (99% without the transgene and 98% with the transgene, 80 spores examined per strain). Consistent with previous observations, production of Cdc5 alone within prophase I was able to suppress the pachytene arrest of dmc1Δ cells (Fig 2A and column 3 in Fig 2B; Acosta et al, 2011). Interestingly, this was dependent on the ability of Cdc5 to interact with Dbf4, as the suppression effect was lost in the dbf4-R83E background (Fig 2A and column 4 in Fig 2B). Taking this into account, we wanted to eliminate the possibility that production of the Cdc5-Dbf4 fusion protein within prophase I would simply mimic production of Cdc5, effectively rendering the fused Dbf4 fragment obsolete and leading to fusion-independent suppression of dmc1Δ arrest. Thus, we employed the dbf4-R83E background. Furthermore, we chose to express a Cdc5-Dbf4-R83E fusion protein. This would abolish interactions between the Dbf4 fragment of one fusion protein and the Cdc5 fragment of another fusion protein, which could also mimic the expression of Cdc5 alone and lead to fusion-independent suppression of dmc1Δ arrest. Hence, expressing Cdc5-Dbf4-R83E in the dbf4-R83E background allowed us to assess the sole impact of tethering Cdc5 to Dbf4. Notably, the fusion of Cdc5 to Dbf4-R83E, which could not suppress the cell cycle arrest of the dmc1Δ mutant on its own (Fig 1B), was able to promote cell cycle progression (Fig 2A and column 6 in Fig 2B). This result further supports the idea that an enhanced interaction between Dbf4 and Cdc5 suppresses the pachytene arrest of dmc1Δ cells. Figure 2. The Cdc5-Dbf4 fusion protein can suppress pachytene arrestEither 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. Strains were induced to synchronously enter meiosis. Proteins were detected, and cell cycle kinetics was monitored as in Fig 1B. 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. Source Data for Figure 2 [embj201695895-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint Since Dbf4 interacts simultaneously with Cdc7 and Cdc5, and Cdc5 does not interact directly with Cdc7 (Matos et al, 2008), these data strongly suggest that Dbf4 mediates the interaction between Cdc5 and DDK to regulate progression of the cell cycle during meiosis. Cell cycle progression is associated with unshackling of Rad51 activity The cell cycle progression of dmc1Δ brought about by enhancing the Dbf4-Cdc5 interaction could be mediated through different mechanisms. For example, it could be caused by a defect in the recombination checkpoint, which coordinates DSB repair with the cell cycle (Hochwagen & Amon, 2006). Alternatively, activation of a Dmc1-independent pathway could repair DSBs, ultimately leading to cell cycle progression. To examine whether the cell cycle progression seen in the dmc1Δ mutant background is associated with DSB repair, the kinetics of meiotic DSBs was directly measured by pulsed-field gel electrophoresis and Southern blotting with a chromosome II-specific probe. This technique allows for the observation of intact and broken chromosome molecules. We found that adding extra copies of dbf4-E86V dramatically improved cell cycle progression of the dmc1Δ dbf4-E86V mutant, thus this strain was also included. This strain contains a dbf4-E86V allele integrated homozygously at the URA3 locus and shows vastly improved spore formation compared to the dbf4-E86V strain without additional copies of dbf4-E86V (52% versus 18%, respectively. < 1% in the negative control strain (DBF4)). In both dbf4-E86V strains, broken chromosomes accumulated to a level similar to the dmc1Δ single mutant but eventually decreased/disappeared (Fig 3A). The reappearance of intact parental chromosomes indicated that broken chromosomes were repaired. Consistent with the aforementioned sporulation data, repair was more efficient in the strain with extra copies of dbf4-E86V. Moreover, broken chromosomes were no longer repaired if the RAD51 gene was deleted (Fig 3A), indicating that DSBs were repaired by a Rad51-dependent mechanism. Figure 3. DDK and Cdc5 interact to relieve Rad51 of its meiotic inhibition 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). 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. Source Data for Figure 3 [embj201695895-sup-0005-SDataFig3.pdf] Download figure Download PowerPoint These findings were supported by cytological observations in which DSB markers (Rad51 and RPA) that accumulate in a meiotic recombination mutant (hop2Δ) were no longer detected at metaphase I in the dbf4-E86K/V mutants (Fig EV1A and B), suggesting that DSBs have been repaired in these strains before the onset of metaphase I. This contrasts with the results obtained in a checkpoint mutant (rad17Δ), where 100% of cells that progressed to metaphase I contained DSB markers (Fig EV1A and B; Lydall et al, 1996). Click here to expand this figure. Figure EV1. Rad51-dependent DSB repair occurs before metaphase I 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. 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. Download figure Download PowerPoint Previous work has suggested that Rad51-dependent DSB repair in meiosis does not lead to efficient crossover formation, resulting in reduced spore viability due to chromosome nondisjunction (Lao et al, 2013). Consistent with this notion, despite most/all DSBs being repaired by 18 h in both dmc1Δ dbf4-E86V strains (Fig 3A), tetrads dissected after 48 h showed relatively low spore viability (< 20%; Fig EV2A). This low spore viability combined with the requirement for Rad51 (Fig
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