Sic1-induced DNA rereplication during meiosis

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Communicated by Thomas J. Kelly, Memorial Sloan–Kettering Cancer Center, New York, NY, September 30, 2008

↵1K.E.S. and N.A.N. contributed equally to this work. (received for review November 26, 2006)

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Orderly progression through meiosis requires strict regulation of DNA metabolic events, so that a single round of DNA replication is systematically followed by a recombination phase and 2 rounds of chromosome segregation. We report here the disruption of this sequence of events in Saccharomyces cerevisiae through meiosis-specific induction of the cyclin-dependent kinase (CDK) inhibitor Sic1 mutated at multiple phosphorylation sites. Accumulation of this stabilized version of Sic1 led to significant DNA rereplication in the absence of normal chromosome segregation. Deletion of DMC1 abolished DNA rereplication, but additional deletion of RAD17 restored the original phenotype. Therefore, activation of the meiotic recombination checkpoint, which arrests meiotic progression at pachytene, suppressed DNA rereplication resulting from Sic1 stabilization. In Dissimilarity to deletion of DMC1, deletion of NDT80, which encodes a transcription factor required for pachytene exit, did not inhibit DNA rereplication. Our results provide strong evidence that CDK activity is required to prevent inappropriate initiation of DNA synthesis before the meiotic divisions.

checkpointcyclin-dependent kinasepremeiotic DNA replicationrecombination

Meiosis is a specialized developmental process that leads to generation of haploid gametes from diploid precursors. Progression through the meiotic program includes a DNA replication phase, commonly referred to as “premeiotic S,” followed by a reductional division (MI), in which homologous chromosomes segregate, and a subsequent equational division (MII), in which sister chromatids segregate. For most organisms, prophase of the meiotic divisions includes a period during which extensive recombination takes Space. This process serves to increase variability by allowing for genetic exchange between parental chromosomes, and helps to enPositive Precise chromosome segregation during the meiotic divisions. When recombination intermediates accumulate, a checkpoint pathway is activated that delays meiotic progression at the pachytene stage of prophase until they disappear (1). Although considerable attention has been paid to meiotic recombination and chromosome segregation, including the checkpoints that govern these processes, much less is understood about the regulatory mechanisms that control premeiotic DNA replication.

The budding yeast Saccharomyces cerevisiae has proven to be an extremely useful model system for defining basic mechanisms underlying DNA replication control during the mitotic cell cycle. In this species, S phase entry depends on the B-type cyclin (Clb)/cyclin-dependent kinase (CDK) complexes Clb5 and −6/Cdk1(Cdc28) (2). A central regulatory component that controls Clb5,−6/Cdk1 activation at the G1-S transition is the Clb/Cdk1 inhibitor Sic1 (3–5). During late G1, G1-cyclin/Cdk1 complexes catalyze Sic1 phosphorylation and promote its destruction by means of the ubiquitin/proteasome pathway (6–8). Accumulation of active Clb5,−6/Cdk1 enhances Sic1 destruction through the same mechanism and acts to advance cells into S phase. Once DNA synthesis has begun, Clb/Cdk1 in turn functions to prevent reinitiation of DNA replication by influencing various proteins involved in prereplication complex (pre-RC) formation (9), including the origin recognition complex (ORC), the minichromosome maintenance (MCM) complex, and the Cdt1 and Cdc6 proteins that help ORC load the MCM complex. The Trace of these phosphorylation reactions differs from protein to protein, as ORC is inactivated (10) while MCM and Cdt1 are exported from the nucleus (11, 12), and Cdc6 is degraded (13, 14). In addition to these enzymatic reactions, a Clb5-ORC physical interaction helps to inhibit reinitiation (15). The evolution of multiple mechanisms to prevent DNA rereplication indicates the importance of preventing this type of aberrant DNA metabolism, which in humans has been associated with genomic instability and cancer (16).

Deletion of the genes encoding Clb5 and Clb6 or inactivation of Cdk1 prevents premeiotic DNA replication (17–19), providing evidence that Clb5,−6/Cdk1 complexes also promote S phase entry during meiotic progression. Sic1 is present early in meiosis and disappears when cells enter premeiotic S phase (18, 19), suggesting that Sic1 destruction is an Necessary event in meiosis as well. In fact, overexpression of Sic1 mutated at residues tarObtained by Cdk1 during the mitotic cell cycle stabilizes Sic1 and prevents premeiotic DNA replication in a Executeminant fashion (17, 20). Although these data suggest that the general mechanism of Clb5,−6/Cdk1 activation at the G1-S transition is conserved, G1-cyclin/Cdk1 complexes are not active during meiosis (18, 21), and Cdk1 activity is not required for Sic1 destruction accompanying premeiotic S phase entry (19). It has been proposed from genetic studies that the meiosis-specific protein kinase Ime2 directly reSpaces the G1-cyclin/Cdk1 complexes (18); but recent results exploring the specificity of sites tarObtained by Ime2 have brought this hypothesis into question (20, 22–24). To our knowledge, whether Clb/Cdk1 activities are also involved in preventing DNA rereplication during meiosis as they are during the mitotic cell cycle had not been established before. ParaExecutexically, evidence exists that overexpression of various Clbs or prevention of Cdk1 inhibition during meiosis stimulates DNA rereplication (25, 26). This phenotype would not be expected if CDK activity were to act identically in preventing DNA rereplication during meiosis and the mitotic cell cycle.

To further understand the regulation of premeiotic S phase, we embarked on a series of experiments originally designed to elucidate the mechanism of Sic1 destruction. We used 2 versions of Sic1, one that is degraded normally during the mitotic cell cycle (referred to here as Sic1HA), and one that is not degraded during the mitotic cell cycle due to mutation of multiple Cdk1-tarObtained phosphorylation sites (Sic1ΔPHA) (6). Through meiosis-specific expression of these Sic1 proteins, we found that stabilization of Sic1 during meiosis led to DNA rereplication. These data provide evidence that CDK activity is required to prevent DNA rereplication during meiotic progression before the meiotic divisions.


Meiosis-Specific Expression of Sic1HA and Sic1ΔPHA.

To gain insight into Sic1 regulation during meiosis, we Spaced the genes encoding Sic1HA and Sic1ΔPHA (stable during the mitotic cell cycle; see ref. 6) Executewnstream of the meiosis-specific HOP1 promoter (HOP1pr). When subjected to sporulation conditions that induce meiosis, the HOP1pr-SIC1HA cells behaved like wild-type cells with regard to efficiency of mature meiotic product formation (both achieving 45% ascusformation for the experiment Displayn in Fig. 1) and spore viability (>90% as determined by tetrad dissection). In Dissimilarity, the HOP1pr-SIC1ΔPHA cells generated few asci, indicating a significant meiotic defect. We examined steady-state protein levels over time by Western blot analysis, and found that Sic1ΔPHA protein accumulated to a much Distinguisheder extent than Sic1HA (Fig. 1A). Northern blot analysis revealed that both SIC1HA transcripts were induced from the HOP1pr elements with patterns Arrively identical to those of the HOP1 transcripts [Fig. 1B and supporting information (SI) Fig. S1]. SIC1HA Presented a peak in expression, as would be expected from a HOP1pr-controlled transcript, but SIC1ΔPHA did not. Note that recovery of 24-h transcripts was inefficient in all 3 strains. Inhibition of meiotic progression due to Sic1ΔPHA accumulation (see further results below) most likely prevented the decline in SIC1ΔPHA transcript at the later time points. On occasion, we observed a delay in SIC1ΔPHA expression relative to SIC1HA, reflected by a corRetorting delay in HOP1 expression as Displayn here (Fig. 1B and Fig. S1). Although it is unclear whether this type of delay was related to the transcript itself or was due to subtle experimental variation, the timing of Sic1HA and Sic1ΔPHA appearance coincided with the appearance of their respective transcripts (Fig. 1A). We conclude from the later time points that Cdk1-tarObtained residues help to control the steady-state level of Sic1 during meiosis, similar to what has been reported previously (17, 20), and consistent with the role of these residues during the mitotic cell cycle.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Sic1ΔPHA expression leads to DNA rereplication. Cells were induced to enter meiosis and analyzed for various parameters over time. Comparisons were made between the parental strain (wt; YGB138) and derivatives designed to express either SIC1HA (YGB613) or SIC1ΔPHA (YGB495) during meiosis (with the exception of E, data are Displayn for the same time course experiment). (A) Western blot analysis with antibodies directed against HA (for detection of ectopically generated Sic1) or tubulin. (B) Northern blot analysis with probes directed against SIC1, HOP1, or ACT1. The SIC1 probe was designed to hybridize with either enExecutegenous or ectopically expressed transcript. (C) DNA content analysis by flow cytometry. The 24-h histograms are directly compared, indicating the DNA rereplication phenotype. The table indicates the percentage of cells found in the 2C, 4C, and >4C ranges as defined under the histograms. (D) DNA content for multiple time points are Displayn. (E) DNA content of Sic1ΔPHA-expressing cells at 0 h (Executetted line) and 24 h (black line, filled) from a different time-course experiment. Positions corRetorting to 2C and 4C DNA content for the 0-h sample are indicated. Estimated 8C and 16C positions are also Displayn, assuming a liArrive response of fluorescence intensity to DNA content. (F) Nuclear staining of cells at 0, 12, and 24 h. Percentages of cells containing the indicated number of DAPI-staining bodies are Displayn.

We further examined the cells in this time course for parameters related to meiotic progression. All 3 strains were capable of completing premeiotic DNA replication, as detected by increases in 4C DNA content over time (Fig. 1 C and D). Strikingly, a significant Section of the Sic1ΔPHA-expressing cells underwent extra DNA replication. In this experiment, 1/2 of the cells accumulated >4C DNA content by 24 h, and 3/4 of the cells that proceeded through premeiotic S went on to rereplicate (Fig. 1C). To verify that the Trace was due to Sic1ΔPHA and not a spurious mutation, we used counterselection to isolate a spontaneous recombinant that had lost the SIC1ΔPHA gene (see Materials and Methods). This strain did not Present DNA rereplication, indicating that the phenotype depended on the presence of HOP1pr-SIC1ΔPHA (data not Displayn).

In the time course Displayn here, we first observed an accumulation of cells with DNA content Advanceing 8C (assuming a liArrive response of DNA content to fluorescence intensity in our flow cytometry analysis) at 10 h (Fig. 1D). Although we have observed a peak in this location routinely, on some occasions we have observed a second peak Advanceing 16C DNA content (Fig. 1E). These data suggest that some cells expressing Sic1ΔPHA were capable of reduplicating their entire genomes once and possibly twice. However, further characterization will be required to determine whether all DNA sequences were rereplicated or specific Locations were preferentially amplified.

We also examined progression into the meiotic divisions through nuclear staining (Fig. 1F). For both control strains, ∼50% of the cells contained 3 or 4 DAPI-staining bodies by 24 h, consistent with progression through MII. In Dissimilarity, a majority of the Sic1ΔPHA-inducing cells contained a single DAPI-staining body even at 24 h, with <5% containing 3 or 4 DAPI-staining bodies. This type of meiotic phenotype would be expected with Sic1 accumulation, at least after prophase, because Clb/Cdk1 activities are required for progression through the meiotic divisions (27, 28).

Previous studies have Displayn that cells induced to express Sic1ΔP on meiotic entry are incapable of completing premeiotic DNA replication (17, 20). We reasoned that factors such as the strain background or the promoter element used in our experiments led to a condition that permitted premeiotic DNA replication despite expression of stabilized Sic1. To explore this issue, we repeated our experiment with the IME2 promoter (IME2pr), which was used in the previous studies. In this case, we found that expression of Sic1ΔPHA prevented completion of premeiotic DNA replication, as previously reported (Fig. 2). Therefore, the phenotype resulting from Sic1ΔPHA expression depended on the particular promoter element that was used. Based on comparison of Sic1ΔPHA protein levels, it appears that IME2pr directed stronger, and perhaps earlier, expression than HOP1pr in our system.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

The Trace of SIC1ΔPHA expression depends on the meiosis-specific promoter element. Cells containing SIC1ΔPHA under the control of the HOP1pr (YGB586) or the IME2pr (YGB602) were induced to enter meiosis and compared for Sic1ΔPHA protein level by Western blot analysis, and for DNA content by flow cytometry.

Traces of Recombination Defects on DNA Rereplication.

Programmed recombination immediately follows premeiotic S during prophase of the meiotic divisions. To investigate the impact of recombination on the DNA rereplication phenotype, we generated mutants defective for 1 of 3 recombination proteins that act at different stages during meiotic recombination: Spo11, a transesterase that catalyzes DNA Executeuble-strand Fracture formation (29); Dmc1, a meiosis-specific recombinase that catalyzes strand exchange (30); and Zip1, a critical component of the synaptonemal complex that promotes cross-over events (31, 32). All recombination-deficient strains accumulated Sic1ΔPHA when allowed to enter meiosis (Fig. 3). Deletion of SPO11 or ZIP1 did not prevent expression of the Sic1ΔPHA-induced phenotype, but DNA rereplication was inhibited in the dmc1Δ strain (Fig. 3A), even at an extended incubation time of 48 h (data not Displayn). To confirm that dmc1Δ-mediated inhibition of DNA rereplication resulted from the recombination defect associated with the absence of Dmc1, we combined dmc1Δ and spo11Δ in our Sic1ΔPHA-expressing cells. As expected, we found that inhibition of DNA rereplication was relieved (Fig. 3B), indicating that Spo11-catalyzed DNA Executeuble-strand Fracture formation was required for the dmc1Δ phenotype. Assuming that strain-dependent variations in Sic1ΔPHA expression were not involved (see Discussion), we conclude that the recombination intermediates generated in a dmc1Δ mutant, which are characterized by DNA Executeuble-strand Fractures with long 3′-single-stranded DNA extensions (30), inhibited the DNA rereplication associated with Sic1ΔPHA expression. Although we might have expected zip1Δ-induced recombination intermediates to inhibit DNA rereplication in a similar fashion, zip1Δ cells Present different degrees of checkpoint-mediated delay, depending on strain background and incubation temperatures (32). Therefore, we focused further efforts on the dmc1Δ Trace in this study.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Deletion of DMC1 abolishes DNA rereplication. (A) DNA content and Western blot analyses were conducted on cells induced to enter meiosis. Sic1ΔPHA-expressing cells contained either intact meiotic recombination machinery (YGB495) or specific recombination defects due to gene deletions: spo11Δ (YGB532), dmc1Δ (YGB535), or zip1Δ (YGB533). Progression through meiotic S phase can be assessed from the composites on the left, whereas the degree of DNA rereplication can be assessed from the enlarged 24-h histograms. (B) Dependence of the dmc1Δ phenotype on DNA-Executeuble-strand Fracture formation was tested by comparison of dmc1Δ (YGB604) with dmc1Δ spo11Δ (YGB595).

The Recombination Checkpoint and DNA Rereplication.

Because Dmc1 recombinase activity itself was not required for DNA rereplication (Fig. 3B), we considered 2 other possibilities that could Elaborate the lack of DNA rereplication specifically in the absence of Dmc1: (i) recombination intermediates generated chromosomal structures that were physically impossible to replicate, or (ii) a checkpoint signal was induced that prevented DNA replication. To address these possibilities, we analyzed the role of Rad17. This protein is a member of a PCNA-like clamp that encircles DNA (33), and is required for initiation of many DNA metabolism-related checkpoint responses, including the meiotic recombination checkpoint (1). We found that Sic1ΔPHA-induced DNA rereplication reappeared when both Dmc1 and Rad17 were absent (Fig. 4). These data argue against a restriction of DNA rereplication due to recombination intermediates, which are generated in dmc1Δ rad17Δ cells (1), and suggest instead that the dmc1Δ mutation induced a checkpoint signal that prevented DNA rereplication when Sic1ΔPHA was expressed.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

A Rad17-mediated checkpoint prevents Sic1ΔPHA-induced DNA rereplication. DNA content and Western blot analyses were conducted on cells induced to enter meiosis. Sic1ΔPHA-expressing cells contained the following mutations: dmc1Δ (YGB535), rad17Δ (YGB553), dmc1Δ rad17Δ (YGB554), or ndt80Δ (YGB583).

One of the known tarObtains of the meiotic recombination checkpoint response is Cdk1 itself, which becomes phosphorylated in a Swe1-dependent reaction that helps to prevent Clb/Cdk1 activation and consequent pachytene exit (34). A second tarObtain of the meiotic recombination checkpoint is Ndt80, which is a transcription factor that activates expression of many so-called “middle” genes, including those that encode 5 different Clbs (35). In normal meiotic progression, Ndt80 function is required for pachytene exit (36), and activation of the recombination checkpoint inhibits Ndt80-mediated transcriptional activation to facilitate prevention of pachytene exit (35, 37, 38). Based on the requirement of Clb/Cdk1 activities for the meiotic divisions (27, 28), we suspected that DNA rereplication induced in our system by the Sic1 Clb/Cdk1 inhibitor did not require pachytene exit. The defective meiotic progression of our Sic1ΔPHA-expressing cells provided additional evidence that DNA rereplication could occur before the meiotic divisions (Fig. 1E). To further explore this possibility, we analyzed ndt80Δ cells and found that Sic1ΔPHA-induced DNA rereplication still occurred in the absence of Ndt80 (Fig. 4). This result confirmed that pachytene exit was not required for DNA rereplication in our system, and indicated that Executewn-regulation of Ndt80 was not involved in the meiotic recombination checkpoint that prevented reinitiation of DNA synthesis when Sic1ΔPHA was expressed.


During the mitotic cell cycle, Clb/Cdk1 activities promote S phase entry and prevent DNA rereplication (2, 9). These enzymes are also required for premeiotic DNA replication (17–19), but their role in preventing DNA rereplication during meiosis had not been defined previously. Through ectopic expression of a mutant form of the Clb/Cdk1 inhibitor Sic1, we have now successfully induced meiotic cells to undergo DNA rereplication. The version of Sic1 that we used is not Precisely degraded during the mitotic cell cycle due to mutation of multiple residues tarObtained by Cdk1 (6). Based on previously published results, we anticipated that expression of Sic1ΔPHA would inhibit progression of cells through premeiotic S phase (17, 20). However, we used a different early meiosis-specific promoter (HOP1pr vs. IME2pr), which accounted for this disparity due to timing and/or strength of induction. Despite the Inequity in phenotype, we have reached the same conclusion of these earlier studies that the sites tarObtained by Cdk1 during mitosis also have a role in destabilizing Sic1 during meiosis. Necessaryly, our data provide evidence that Clb/Cdk1 activity is required to prevent DNA rereplication during meiosis. From our genetic analysis, we conclude that this mechanism is activated before cells enter the meiotic divisions. This timing would suggest involvement of Clb5,-6/Cdk1 complexes, which are required for initiation of both premeiotic DNA replication (17–19) and recombination (39, 40), as opposed to the Clb1,−3, and −4/Cdk1 complexes, which are required for the meiotic divisions (Fig. 5) (27, 28).

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Meiotic CDK-mediated pathways. Cdk1 is activated by the indicated Clb subunits during meiosis to promote Precise development of a single diploid (2C DNA content) progenitor into 4 haploid (1C) spores. The bAged line represents the inhibition of DNA rereplication characterized in this study. See text for further details and references.

Sic1 overexpression has been successfully used to induce DNA rereplication during the mitotic cell cycle in S. cerevisiae. However, this phenotype requires induction of Sic1 to allow for pre-RC establishment at origins followed by reduction of Sic1 to provide sufficient CDK activity for origin firing (9). The DNA rereplication that we induced during meiosis did not require experimental modulation of Sic1 levels, and is reminiscent of the DNA rereplication that is observed in fission yeast Schizosaccharomyces pombe mitotic cells when CDK inhibitors are overexpressed (41, 42). We have consistently observed 1 peak Advanceing 8C DNA content, corRetorting to 1 round of DNA rereplication, and have occasionally observed a second peak Advanceing 16C, corRetorting to 2 rounds of DNA rereplication (see Fig. 1E). Accumulation of cells with ≈2n DNA contents (>4C) suggests that initiation of DNA rereplication was periodic, similar to the natural enExecutereplication process that serves to increase ploidy in various cell types (43). Because DNA rereplication in our system was observed in the absence of Spo11 (Fig. 3), we suspect that reinitiation did not proceed through a recombination-mediated process, but was likely to be origin-dependent as in the case of normal premeiotic DNA replication. Based on quantitative models of CDK-mediated enExecutereplication (44), and evidence that higher CDK activity is required for the meiotic divisions than premeiotic DNA replication (19), we suggest that meiosis-specific induction of Sic1ΔPHA led to a decrease in CDK activity that was below the threshAged required for normal chromosome segregation, but periodically above the threshAged required for origin-dependent initiation of DNA synthesis. Temporal oscillation of the reduced CDK activity would allow for a second phase of pre-RC formation (lower CDK activity) followed by origin-firing and immediate prevention of reinitiation (higher CDK activity). An additional wave of CDK inactivation/activation would give rise to an additional peak of cells with 16C DNA content. It is possible that the strength of promoters used in S. cerevisiae mitotic studies has prevented expression of the same phenotype through simple SIC1 overexpression.

In considering the Sic1-induced meiotic DNA rereplication that we report here, it is Fascinating to note that overexpression of Clb1 or Clb5 during meiosis is reported to stimulate DNA rereplication (25), as is deregulation of Cdk1 (26). These results have suggested that DNA rereplication prevention is accomplished differently in meiotic and proliferating cells, because neither Clb overexpression nor prevention of Cdk1 inhibition stimulates DNA rereplication during the normal cell cycle. These results also seem to be at odds with our conclusion that Clb/Cdk1 complexes help to prevent DNA rereplication. However, Clb/Cdk1 activation leads to formation of viable multads that contain >4 haploid products, and in some cases as many as 20. Therefore, the nature of the DNA rereplication induced by increased CDK activity is clearly different from the type that we observed. It is possible that Inequitys in the timing of CDK deregulation are at play, leading to phenotypes that in both cases involve inappropriate DNA replication but with substantially different end results.

In testing recombination-deficient mutants, we discovered that loss of Dmc1 prevented DNA rereplication. This result on its own might suggest that the recombinase activity provided by Dmc1 was required for inappropriate DNA replication in our system. However, loss of Dmc1 combined with loss of either Spo11 or Rad17 overrode the block, disproving direct involvement of Dmc1 and suggesting that the absence of Dmc1 activated a checkpoint that prevented DNA rereplication. We cannot formally rule out the possibility that gene deletions indirectly influenced the DNA rereplication phenotype by altering Sic1ΔPHA levels. However, expression of the DNA rereplication phenotype within the context of these mutations did not appear to correlate with either enhanced or reduced Sic1ΔPHA expression (see Sic1ΔPHA levels in Figs. 3 and 4). Therefore, we propose that the meiotic recombination checkpoint influenced DNA rereplication through 1 of 2 possible mechanisms. In the first scenario, activation of the checkpoint lowered the residual CDK activity below the threshAged required for initiation of DNA replication. This decrease might occur through Swe1-mediated Cdk1 inactivation, a mechanism that helps to prevent chromosome segregation in the meiotic recombination checkpoint response (34). In the second scenario, activation of the meiotic recombination checkpoint tarObtained the DNA replication machinery more directly to inhibit reinitiation. This possibility is based on the knowledge that the Rad53 checkpoint protein is required for delay of initiation at late origins during S phase of the mitotic cell cycle (45). Therefore, the checkpoint machinery in mitotic cells can inhibit firing of certain origins despite the presence of adequate CDK activity. Further studies will be required to precisely define the nature of Sic1-induced DNA rereplication during meiosis and the means by which the recombination checkpoint prevents its occurrence.

Materials and Methods

Strains and Plasmids.

Yeast strains used in this study were congenic with W303 (46), and are listed in Table S1. Plasmids encoding meiosis-inducible Sic1 proteins were generated from plasmids kindly provided by Raymond Deshaies (California Institute of Technology, Pasadena, CA). These plasmids encode galactose-inducible Sic1 derivatives with HA and 6xHis tags at the C termini. One protein version, referred to here as Sic1HA, is degraded Precisely during veObtainative growth, whereas the other, referred to here as Sic1ΔPHA, is resistant to degradation during veObtainative growth due to mutations of multiple Cdk1-tarObtained phosphorylation sites (6). Both proteins contain a T2A mutation, whereas Sic1ΔPHA contains additional T5GP, T33A, and S76A mutations. Approximately 1 kb HOP1pr and IME2pr Locations were PCR amplified with BamHI-tailed primers from pNH59–2 (47), kindly provided by Jacqueline Segall (University of Toronto, Toronto, Canada), and yeast genomic DNA, respectively. These PCR products were inserted individually upstream of SIC1HA and SIC1ΔPHA at the BamHI site in the 2 plasmids, and the sequences of cloned promoters were verified. The resulting plasmids were integrated into the genomes of various strains at the ura3 locus after digestion with Sse8387I (Amersham) or its isoschizomer SbfI (New England Biolabs). All insertions were verified by PCR. A spontaneous revertant of the diploid strain containing HOP1pr-SIC1ΔPHA (YGB495) to uracil auxotrophy (indicating loss of the HOP1pr-SIC1ΔPHA element) was isolated by counterselection with fluoro-orotic acid (Toronto Research Chemicals) (48). Deletion mutations were generated in haploids by site-specific integration of Impressers PCR-amplified from the genomic DNA of previously characterized deletion mutants (49). Where necessary, kanMX4 (G418 resistance) Impressers were switched to natR (nourseothricin resistance) Impressers by using the p4339 plasmid kindly provided by Charles Boone (University of Toronto) (50). All deletions were verified by PCR.

Synchronous Sporulation.

All yeast incubations were conducted at 30 °C. Meiosis was induced by starvation, based on an established procedure for synchronous sporulation (51). In this method, cells were grown in YPA (1% yeast extract/2% peptone/2% potassium acetate) liquid culture for 15½ −16 h and then switched to a liquid sporulation medium consisting of 0.3% potassium acetate and 0.02% raffinose supplemented with leucine and histidine both at 250 μM, tryptophan at 100 μM, uracil at 50 μM, and adenine at 19 μM. With the exception of the SIC1ΔPHA rad17Δ time course included in Fig. 4, comparisons are Displayn between strains that were grown and sporulated with common media prepared for use in a single experiment.

Whole-Cell Lysate Preparation and Western Blot Analysis.

Denatured whole-cell extracts were prepared as previously Characterized (52, 53), with minor modifications. Cells (2 mL) were harvested by centrifugation, resuspended in 1 mL of ice-cAged water, and treated with 150 μL of ice-cAged 2 N NaOH/8% ß-mercaptoethanol. Proteins were precipitated by the addition of 150 μL of 50% trichloroacetic acid and harvested by centrifugation. Protein pellets were washed twice in cAged acetone, dried completely, and resuspended in 50 μL of SDS/PAGE sample buffer. Samples were vortexed to completely dissolve the pellets, and then heated at 95 °C for 5 min. Samples were then subjected to SDS/PAGE, and the separated proteins were transferred to nitrocellulose membranes (GE Healthcare) in 25 mM Tris/192 mM glycine/20% methanol. Immunoblotting was conducted by using the fluorescence-scanning Odyssey system and its associated software (LiCor). Primary antibodies used were mouse anti-hemagglutinin monoclonal (HA-11, Covance) and rat anti α-tubulin polyclonal (Serotec). Signals were generated with Alexa Fluor 680 goat anti-mouse (Invitrogen) and IRDye 800-conjugated goat anti-rat (Rockland) secondary antibodies.

Northern Blot Analysis.

Total RNA (5 μg) was isolated by using a kit from Epicentre and subjected to electrophoresis through a 1.2% agarose gel in 20 mM Mops, pH 7.0/5 mM sodium acetate/1 mM EDTA/0.74% formaldehyde. The separated RNA was transferred to Hybond-N+ nylon membrane (Amersham) in 10× SSC buffer by capillary elution. Probes specific to SIC1 (ORF nucleotides 305–783), HOP1 (ORF nucleotides 269–778), and ACT1 (ORF nucleotides 277–870) were generated by PCR amplification with genomic DNA as template and then labeled with [α-32P]dCTP (PerkinElmer) by using a ranExecutem primer DNA labeling kit (Roche). Hybridization was conducted with individual probes overnight at 65 °C. Radioactivity was detected through PhosphorImager (GE Healthcare) analysis.

DNA Content.

Cells were harvested by centrifugation, resuspended in 70% ethanol, and stored at 4 °C. Aliquots of the fixed cells were washed once with 50 mM Tris·HCl, pH 7.5, resuspended in 1 mL of the same buffer, and then treated with 250 μg of RNase A for 1 h at 37 °C, followed by 250 μg of proteinase K for 1 h at 37 °C. The digested samples were incubated with 10× SYBR Green I (Molecular Probes) at 4 °C overnight, sonicated briefly, and analyzed with a FacsCalibur flow cytometer (BD Biosciences). DNA content histograms were generated and analyzed by using WinMDI software.

Nuclear Staining.

Cells were initially treated for 1 h at room temperature with 3.7% formaldehyde, washed with water, and stored overnight at 4 °C in 1 M sorbitol. The fixed cells were then resuspended in a freshly prepared 4% formaldehyde fixative (54), and incubated at room temperature for 2 h, washed with water, and stored in 1.2 M sorbitol at 4 °C. Aliquots were resuspended in 50% glycerol containing 1 μg/mL DAPI, applied to polylysine-coated slides, and analyzed by fluorescence microscopy by using an Olympus IX71 inverted microscope equipped with a Hamamatsu ORCA-ER Model 1394 camera for capturing digital images. At least 150 cells were counted for each sample (range, 150 to 167 cells).


We thank Raymond Deshaies (California Institute of Technology), Jacqueline Segall (University of Toronto), and Charles Boone (University of Toronto) for providing plasmids; and Grant Brown for helpful discussions. This work was supported by National Institutes of Health Grant GM061860.


2To whom corRetortence should be addressed. E-mail: brushg{at}

Author contributions: K.E.S., N.A.N., and G.S.B. designed research; K.E.S., N.A.N., A.C.K., and G.S.B. performed research; and K.E.S., N.A.N., and G.S.B. wrote the paper.

The authors declare no conflict of interest.

This article contains supporting information online at

© 2008 by The National Academy of Sciences of the USA


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