Skip to main content
NCBI home page
UKPMC Funders Author Manuscripts logoLink to UKPMC Funders Author Manuscripts
. Author manuscript; available in PMC: 2013 Jan 31.
Published in final edited form as: Cell. 2011 Sep 30;147(1):158–172. doi: 10.1016/j.cell.2011.08.032

Regulatory Control of the Resolution of DNA Recombination Intermediates during Meiosis and Mitosis

Joao Matos 1,2, Miguel G Blanco 1,2, Sarah Maslen 1, J Mark Skehel 1, Stephen C West 1,*
PMCID: PMC3560330  EMSID: EMS51351  PMID: 21962513

SUMMARY

The efficient and timely resolution of DNA recombination intermediates is essential for bipolar chromosome segregation. Here, we show that the specialized chromosome segregation patterns of meiosis and mitosis, which require the coordination of recombination with cell-cycle progression, are achieved by regulating the timing of activation of two crossover-promoting endonucleases. In yeast meiosis, Mus81-Mms4 and Yen1 are controlled by phosphorylation events that lead to their sequential activation. Mus81-Mms4 is hyperactivated by Cdc5-mediated phosphorylation in meiosis I, generating the crossovers necessary for chromosome segregation. Yen1 is also tightly regulated and is activated in meiosis II to resolve persistent Holliday junctions. In yeast and human mitotic cells, a similar regulatory network restrains these nuclease activities until mitosis, biasing the outcome of recombination toward non-crossover products while also ensuring the elimination of any persistent joint molecules. Mitotic regulation thereby facilitates chromosome segregation while limiting the potential for loss of heterozygosity and sister-chromatid exchanges.

INTRODUCTION

During mitosis and meiosis, cells commit to the transmission of a complete set of chromosomes to the next generation. Whereas the bipolar segregation of replicated sister chromatids keeps the chromosome complement unchanged during mitosis, meiosis generates haploid gametes from diploid germ cells through a single DNA replication phase followed by two consecutive rounds of chromosome segregation. Homologous chromosomes (homologs) segregate in meiosis I and sister chromatids disjoin in meiosis II.

The ability of meiotic cells to segregate homologs during meiosis I requires the coordination of a series of specialized events. Most organisms use reciprocal recombination between maternal and paternal chromatids to create crossovers (COs) that link homologs through cohesin-mediated sister-chromatid cohesion. When sister kinetochores attach to microtubules from the same pole, rather than from opposite poles as occurs in mitosis, the chiasmata enable the meiosis I spindle to pull maternal and paternal centromeres in opposite directions. Therefore, and in contrast to mitosis, the formation of meiotic COs provides the indispensable mechanical basis for accurate chromosome segregation.

The importance of CO formation during meiosis can be appreciated by the complex and potentially deleterious strategy that cells employ in their generation. Most organisms produce COs upon deliberate chromosome breakage, which is initiated by double-strand break (DSB) formation mediated by meiosis-specific expression of Spo11 (Keeney et al., 1997). Recombination with a homologous chromosome leads to the formation of joint molecule (JM) intermediates in which the interacting DNAs are linked by double Holliday junctions (dHJs) (Allers and Lichten, 2001; Hunter and Kleckner, 2001; Schwacha and Kleckner, 1995).

Studies from various organisms indicate that there are at least three pathways by which HJs can be processed to generate COs. In budding yeast these involve the Mus81-Mms4, Slx1-Slx4, and Yen1 endonucleases (Fricke and Brill, 2003; Ip et al., 2008; Kaliraman et al., 2001). Different organisms, however, show a specific dependence on one pathway or another. For example, meiotic CO formation in Schizosaccharomyces pombe is dependent only upon Mus81-Eme1 (Eme1 is the ortholog of Mms4) (Boddy et al., 2001; Osman et al., 2003), and a Yen1 ortholog cannot be identified in its genome (Ip et al., 2008). In contrast, Saccharomyces cerevisiae mus81Δ mutants show a small reduction in CO formation and form spores efficiently, albeit with reduced viability (~50% of wild-type), suggesting that Mus81-Mms4 plays a relatively modest role in HJ processing and CO formation (de los Santos et al., 2001, 2003; Haber and Heyer, 2001). In budding yeast, Slx1-Slx4 appears to be relatively unimportant for CO formation, as meiotic defects are not observed in slx1 or slx4 mutants (Mullen et al., 2001), and the role of Yen1 has not been investigated. However, Yen1 and Mus81-Mms4 provide overlapping functions in promoting JM resolution and CO formation during mitotic DNA repair (Blanco et al., 2010; Ho et al., 2010; Tay and Wu, 2010). These observations highlight the possibility that a degree of functional redundancy between nucleases might obscure their specific contributions toward JM resolution and the completion of meiotic recombination.

The efficient and appropriate resolution of recombination intermediates is a key event in all cells. During meiosis, dHJs need to be resolved to form the COs necessary for the segregation of homologs, whereas in mitotic cells noncrossover (NCO) formation is favored to avoid the potential for loss of heterozygosity and high levels of sister-chromatid exchanges (SCEs). Indeed, during mitotic recombination CO formation is avoided either by the use of antirecombinogenic pathways that disengage JMs at an early stage, or by the actions of enzymes that promote dHJ dissolution. For example, in budding yeast, DNA helicases such as Srs2 and Sgs1 have been shown to suppress CO formation and to play important roles in recombinational DNA repair (Gangloff et al., 1994; Ira et al., 2003).

The timing by which JMs are processed is also critical because unless they are disengaged/processed at the appropriate time, their presence will constitute a physical impediment to chromosome segregation. In budding yeast meiosis, the timing of JM resolution and CO formation is coordinated with cell-cycle progression through the NDT80-dependent expression of the Polo-like kinase Cdc5, to ensure the bipolar segregation of fully resolved DNA (Clyne et al., 2003; Sourirajan and Lichten, 2008). Ndt80-mediated transcription of Cdc5, and other components of the chromosome segregation machinery, is activated in late pachytene as recombination and synapsis check-points are satisfied and cells prepare for meiosis I (Chu et al., 1998). Interestingly, whereas Cdc5-depleted cells accumulate JMs, such as dHJs, they are still capable of expressing NDT80-dependent genes and assemble meiosis I spindles (Clyne et al., 2003; Lee and Amon, 2003). These observations indicate that the formation/resolution of late recombination intermediates might not be under checkpoint surveillance. Because a single unmonitored JM could result in chromosome nondisjunction and aneuploidy, cells need to eliminate dHJs prior to chromosome segregation, and it is possible that Cdc5 kinase may play a role in regulating the timing of such events.

In this work, we have analyzed the activities of cellular Holliday junction resolvases, in both yeast and human cells. We find that the activities of yeast Mus81-Mms4 (human MUS81-EME1) and Yen1 (GEN1) are tightly regulated throughout the cell cycle, in both meiotic and mitotic cells, in order to coordinate JM resolution with chromosome segregation. Regulation is mediated through cycles of phosphorylation/dephosphorylation that directly modulate HJ resolvase activity, and in the case of Mus81-Mms4 these regulatory cycles are dependent upon Cdc5. These results bring a new cell-cycle dimension to the completion of homologous recombination in both meiotic and mitotic cells. Moreover, the combination of tight activity regulation with multiple overlapping activities ensures the elimination of JMs, while also providing a flexible control that determines the outcome (CO versus NCO) of the recombination process according to the needs of meiotic or mitotic division.

RESULTS

Mus81-Mms4 and Yen1 Ensure the Completion of Meiotic Recombination

To determine the roles and potential interplay between Mus81-Mms4, Yen1, and Slx1-Slx4 during meiotic recombination, S. cerevisiae diploids were generated with deletions in one or more of the corresponding genes. The yen1Δ, slx1Δ, and slx4Δ single mutants formed spores with similar efficiency to wild-type cells (>90% sporulation efficiency), whereas mus81Δ exhibited a slightly reduced efficiency (80% sporulation) (Figure 1A). The defect in mus81Δ mutants was enhanced by the lack of YEN1, as only ~30% of the mus81 Δ yen1Δ cells formed mature spores. In contrast, other double mutant combinations, such as mus81 Δ slx1Δ, mus81 Δ slx4Δ, yen1Δ slx1Δ, or yen1Δ slx4Δ failed to show a synthetic effect in terms of sporulation efficiency.

Figure 1. Requirement for Mus81-Mms4 and Yen1 in Meiosis.

Figure 1

Open in a new tab

(A and B) The efficiencies of spore formation and viability were measured after 24 hr at 30°C with the indicated strains. Bars indicate standard errors.

(C) Representative asci are shown.

Yen1 and Mus81-Mms4 play overlapping roles during DNA repair in proliferating cells (Blanco et al., 2010; Ho et al., 2010; Tay and Wu, 2010). To eliminate the possibility that defects in sporulation in mus81 Δ yen1Δ mutants might result from the accumulation of toxic repair intermediates from the preceding mitosis, cells expressing PCLB2-MMS4 were generated to specifically deplete Mms4 from meiotic cells (Oh et al., 2008). The PCLB2-MMS4 yen1Δ mutants showed a similar defect in sporulation to that of mus81 Δ yen1Δ (Figure 1A) or mms4Δ yen1Δ (see Figure S5C available online). Furthermore, using mus81 Δ yen1Δ spo11Δ strains, we confirmed that this defect was a consequence of incomplete meiotic recombination (Figure 1A).

Measurements of spore viability revealed a complete defect in the formation of viable spores when the YEN1 deletion was combined with mus81Δ, PCLB2-MMS4, or mms4Δ (Figure 1B and Figure S5D). Examination of the resulting asci showed that the DNA from the majority of mus81 Δ yen1Δ cells was concentrated in a single mass (Figure 1C). Deletion of SPO11 alleviated this problem, showing that the DNA segregation defect stems from the accumulation of unresolved recombination intermediates. In contrast, yen1Δ slx1Δ or yen1Δ slx4Δ exhibited wild-type levels of spore viability (Figure 1B).

Distinct Roles for Mus81-Mms4 and Yen1 in JM Resolution in Meiosis I and II

To determine whether Mus81-Mms4 and Yen1 play distinct or redundant functions, the stages of meiotic chromosome segregation were visualized in yen1Δ, mus81Δ, and mus81 Δ yen1Δ mutants. For this, strains were constructed in which both homologs of chromosome V were marked by GFP at URA3 (homozygous URA3-GFP), a Myc18-tagged version of the anaphase inhibitor Pds1 was expressed, and the meiosis-specific cohesin subunit Rec8 was tagged with Ha3.

In wild-type cells, the metaphase I to anaphase I transition is triggered by Pds1 degradation, followed by spindle elongation, segregation of DNA into two masses, and bipolar segregation of homologous URA3 sequences. The binucleates then reaccumulate Pds1 and form a pair of short meiosis II spindles. Next, a second round of Pds1 degradation initiates the metaphase II to anaphase II transition, resulting in the tetrapolar segregation of URA3-GFP and formation of four distinct nuclei (Figure 2A). Whereas the meiotic segregation events in yen1Δ mutants were similar to wild-type (Figure 2A), mus81Δ mutants displayed an unanticipated pattern of chromosome segregation. Although meiosis I spindles were assembled with similar kinetics to control cells, most mus81Δ mutants failed to segregate homologous chromosomes upon Pds1 destruction at anaphase I (Figure 2A and Figure S1). Pds1 reaccumulated normally as meiosis II spindles were formed in mononucleated cells, followed by a second round of Pds1 destruction. At anaphase II, however, sister chromatids segregated efficiently in a tetrapolar fashion, indicating that JM resolution occurred after meiosis I, ultimately allowing chromosome segregation. In contrast, mus81 Δ yen1Δ cells formed meiosis I and II spindles, underwent two cycles of Pds1 accumulation and destruction, but failed to segregate the bulk of their DNA at either anaphase I or anaphase II (Figure 2A and Figure S1). Control experiments showed that this was not due to the persistence of proteinaceous interhomolog connections, as synaptonemal complex (SC) disassembly and cohesin cleavage occurred normally at meiosis I (Figures S1E and S1F). These results indicate that mus81 Δ yen1Δ mutants fail to segregate their chromosomes during meiosis I due to the lack of Mus81-Mms4 activity, and that Yen1 is required for the single meiotic division that takes place in the absence of Mus81-Mms4. The lack of chromosome segregation in the mus81 Δ yen1Δ mutants was due to unresolved meiotic DNA joint molecules, as elimination of DSB formation in mus81 Δ yen1Δ spo11Δ mutants restored both rounds of chromosome segregation (Figure 2B and Figure S1).

Figure 2. Mus81-Mms4 and Yen1 Ensure Chromosome Segregation during Both Meiotic Divisions.

Figure 2

Open in a new tab

(A) Immunofluorescence analysis of meiosis in WT and mutant strains expressing PDS1-myc18 (for securin visualization) and URA3-GFP (GFP marks chromosome V at URA3). Left: images taken at different stages of the cell cycle illustrate chromosome segregation patterns. Right: quantification and kinetics of meiotic progression, indicating the percentage of cells with two DNA masses (two nuclei), those that have undergone the second meiotic division (four nuclei), and those undergoing meiosis I (one spindle) or meiosis II (two spindles).

(B) Determination of the frequency of nuclear division during meiosis I and meiosis II, as described in (A). The percentage of cells at each stage that have undergone the first or second meiotic division (two or four DNA masses) are indicated.

See also Figure S1.

To determine whether Yen1 plays a specific role in the resolution of intersister joint molecules, we analyzed dmc1Δ mek1Δ mutants where interhomolog recombination is suppressed in favor of intersister repair (Niu et al., 2005) and found that YEN1 was dispensable for the segregation of sister chromatids at anaphase II (Figure S1G). Taken together, these results indicate that Yen1 is responsible for the resolution of persistent JMs, possibly as a backup for Mus81-Mms4.

Yen1 Is Tightly Regulated and Activated at the Onset of Anaphase II

Our finding that most mus81Δ mutant cells fail to undergo meiosis I even in the presence of YEN1 indicates that the role of Yen1 in JM resolution is specific for meiosis II. One explanation for this surprising result would be that either Yen1 protein is absent during meiosis I, or that Yen1 activity is kept in check until the onset of meiosis II. We therefore developed a strategy that allowed us to analyze Yen1 activity throughout meiosis using synchronized cells expressing Myc-tagged Yen1 (YEN1-myc9). Following meiotic induction, samples were taken at different time points, Yen1-myc9 was affinity-purified from extracts using anti-Myc beads, and HJ resolvase activity was assayed directly by the addition of synthetic 32P-labeled HJ DNA (see scheme, Figure S2A).

Whereas the levels of Yen1 protein remained relatively constant throughout meiosis, the activity of the protein changed dramatically, as measured by the conversion of HJ DNA into nicked duplex products. Very little HJ resolvase activity was observed from S phase (Figure 3A, lower panel, lanes i and j, and quantification in Figure 3B) until the activity increased sharply as the cells entered meiosis II (Figure 3A, lanes l and m). The precise timing of Yen1 activation was monitored in highly synchronous meiotic cultures using an ndt80Δ arrest/release system, showing that Yen1 activation occurred after the accumulation of the meiosis II-specific cyclin Clb3, at the metaphase II to anaphase II transition (Figure S2B, lanes h–k and Figure S2C). Control anti-Myc immunoaffinity purification from cells expressing either untagged Yen1 (Figure 3A, lanes a–g) or Myc-tagged Yen1-EE (Figure S2D), a catalytic-dead derivative of Yen1 (Ip et al., 2008), showed that Yen1 was directly responsible for the HJ resolution activity.

Figure 3. Activation of Yen1 by Dephosphorylation at the Onset of Meiosis II.

Figure 3

Open in a new tab

(A) Extracts were prepared from WT, YEN1-myc9, and PCLB2-CDC5 YEN1-myc9 strains at 2 hr intervals after transfer into sporulation medium. Yen1-myc9, Cdc5, and Tub2 were detected by western blotting, and Yen1-myc9 was immunoaffinity purified from each extract and assayed for HJ resolution activity. Asterisks indicate 5′-32P-labels.

(B) Quantification of Yen1 HJ resolvase activity relative to the kinetics of meiotic progression as determined from (A).

(C) Kinetics of meiotic progression (spindle morphology and nuclear divisions) and western blot analysis of protein extracts from cells expressing Yen1-myc9. Yen1-myc9 was analyzed by standard or phosphoaffinity (Phos-Tag) SDS-PAGE. Cc: sample from proliferating cells.

(D) Activation of Yen1 by phosphatase treatment. Yen1-myc9 was immunopurified from cells arrested in prophase I using ndt80Δ YEN1-myc9 cells (collected after 6 hr in SPM). Protein fractions were analyzed as in (C) and treated with λ-phosphatase, or inactivated phosphatase, as indicated.

See also Figure S2 and Figure S3.

The human ortholog of Yen1, GEN1, cleaves a variety of DNA substrates including HJs, 5′-flaps, and replication fork structures (Ip et al., 2008; Rass et al., 2010). To determine whether activation of Yen1 might be specific for one particular substrate, we prepared Myc-tagged Yen1 from cells taken at different stages of meiosis and compared their activities using the three DNA substrates. Similar activation profiles were obtained, indicating that the observed regulation most likely operates by a general inhibition of nuclease activity (Figure S2E).

Regulation of Yen1 Activity by Posttranslational Modification

Next, we determined whether the changes to Yen1 activity might be due to posttranslational modifications. When analyzed by phosphoaffinity SDS-PAGE, a method that retards phosphorylated proteins, we observed that the inactivation of Yen1 during the early stages of meiosis correlated with a slow electrophoretic mobility of the protein (Figure 3C, lanes c–e). At the onset of meiosis II, however, the mobility of Yen1 increased significantly (Figure 3C, lanes f and g), and the timing of this event occurred together with the activation of Yen1’s nuclease activity (Figure 3A, lanes k–m). This correlation was confirmed by treating “inactive” Yen1-myc9 (immunoaffinity purified from cells synchronized at prophase I by NDT80 deletion) with λ-phosphatase. Dephosphorylation of Yen1 resulted in a dramatic activation of the nuclease activity of the protein (Figure 3D, lane c), whereas the activity was unaffected by treatment with inactivated λ-phosphatase (Figure 3D, lane d).

These results show that Yen1 activity is directly controlled by its phosphorylation/dephosphorylation status, and provide a mechanistic basis for the modulation of Yen1 activity throughout meiosis. We suggest that the protein is held in an inactive phosphorylated state during DNA replication and meiosis I, favoring JM cleavage by Mus81-Mms4, and is then activated by dephosphorylation as cells undergo the second round of chromosome segregation. These results define a cellular role for Yen1 in meiosis II, in the resolution of persistent JMs prior to segregation, and explain how Yen1 specifically rescues the second meiotic division in mus81Δ mutants.

Slx1-Slx4 Does Not Display HJ Resolvase Activity during Meiosis

In contrast to Mus81-Mms4 and Yen1, the genetic analyses presented in Figure 1 indicate that Slx1-Slx4 plays a very minor role in processing meiotic recombination intermediates. To confirm this, synchronized meiotic cells expressing either Myc-tagged Slx1 or Slx4 were used to carry out immunoaffinity purifications similar to those with Yen1. We were unable to detect Slx1-Slx4 HJ resolvase activity at any stage of meiosis (Figure S3A), whereas resolvase activity was observed in immunoprecipitates from proliferating mitotic cells (Figure S3A, lane cc). These results indicate that Slx1-Slx4 activity might be downregulated in meiosis. We did, however, note that both Slx1 and Slx4 were phosphorylated in meiosis (Figures S3A and S3B), but the consequences of these posttranslational modifications are presently unclear.

Mus81-Mms4 Is Hyperactivated by the Polo-like Kinase Cdc5 at the Onset of Meiosis I

The resolution of interhomolog JMs at the onset of meiosis I depends on the accumulation of Cdc5 (Clyne et al., 2003), but the precise target of Cdc5 in this process remains to be identified. Given that Yen1 and Slx1-Slx4 do not appear to be activated at the time when Cdc5 accumulates (Figure 3A and Figure S3), we determined whether Cdc5 regulates Mus81-Mms4 activity. To do this, Mus81-Mms4, Mms4-myc9 (Figure 4A), or Mus81-myc9 (Figure 5A) were immunoaffinity purified from synchronized meiotic cultures and analyzed for HJ resolution. We observed significant variations in the levels of Mus81-Mms4 activity throughout meiosis. Whereas a basal level of activity was observed at all time points, an increase in activity occurred in meiosis I that was coincident with the expression of Cdc5 (Figure 4A, lanes k and l and Figure 5A, lanes c–e) and the formation of meiosis I spindles (Figures 4A and 4B and Figure 5A). Control experiments, carried out with cells that carried untagged Mms4 (Figure 4A, lanes a–g) or a Myc-tagged catalytically impaired version of Mus81, Mus81-DD (de los Santos et al., 2003) (Figure S4A), confirmed that the HJ resolution activity measured in these experiments was dependent on Mus81-Mms4. PCLB2-CDC5 cells, depleted of Cdc5, formed meiosis I spindles but failed to activate Mus81-Mms4 (Figure 4A, lanes o–u and Figure 4B) whereas they activated Yen1 after a slight delay (Figure 3A, lanes o–u and Figure 3B).

Figure 4. Regulation of Mus81-Mms4 Activity by Cdc5-Dependent Phosphorylation.

Figure 4

Open in a new tab

(A) Upper panels: extracts were prepared from meiotic WT, MMS4-myc9, and PCLB2-CDC5 MMS4-myc9 cells and proteins were detected by western blotting. Lower panel: Mms4-myc9 was immunoaffinity purified and assayed for HJ resolution activity.

(B) Quantification of Mus81-Mms4 activity relative to the kinetics of meiotic progression as determined from (A).

(C) Cdc5 expression in prophase I-arrested cells is sufficient to promote the phosphorylation and activation of Mus81-Mms4. Extracts from ndt80Δ cells expressing Cdc5-ha3 from an estradiol-inducible GAL1 promoter (after 5 hr) were analyzed for the presence of the indicated proteins by western blotting, and immunoaffinity purified Mms4-myc9 was assayed for HJ resolvase activity.

(D) Inactivation of Mus81-Mms4 by dephosphorylation. Mms4-myc9 was immunopurified from cells arrested in metaphase I (PCLB2-CDC20 MMS4-myc9 cells, collected after 8 hr in SPM), treated with λ-phosphatase as indicated, and assayed for HJ resolution activity.

(E) Association of Mms4 with Cdc5. Extracts or anti-Myc immunoprecipitates prepared from PCLB2-CDC20 MMS4-myc9 cells were western blotted for Mms4-myc9 or Cdc5 as indicated.

See also Figure S4.

Figure 5. Hyperactivation of Mus81-Mms4 Ensures Timely JM Resolution and Promotes Chromosome Segregation at Meiosis I.

Figure 5

Open in a new tab

(A) Analysis of Mus81-Mms4 activity from MUS81-myc9 cells expressing either MMS4-WT-ha3 or mms4-14A-ha3, as described for Figure 4.

(B) Phosphorylation map of Mms4. Phosphorylated residues identified by mass spectrometry (red) and predicted CDK consensus sites (blue) are indicated.

(C) Immunofluorescence analysis of meiosis in MMS4-WT YEN1, mms4-14A YEN1, and mms4-14A yen1Δ, as for Figure 2.

(D) Representative asci from the indicated strains.

(E) Cdc5 activates Mus81-Mms4 to promote JM resolution. Physical analysis of recombination at the HIS4LEU2 locus in ndt80Δ cells expressing CDC5-GFP from an estradiol-inducible promoter. Southern analysis of psoralen-crosslinked DNA prepared from meiotic time courses of MMS4, mms4Δ, or mms4-14A strains. The dynamics of JM accumulation and resolution, in the presence or absence of estradiol after 7 hr in SPM, are shown. Protein extracts were analyzed for the presence of the indicated proteins. mc-JM: multichromatid joint molecules; dHJ-JM: double Holliday junction joint molecules.

See also Figure S5, Figure S6, and Table S1.

Consistent with the concept that Cdc5 is involved in the hyperactivation of Mus81-Mms4, we found that Mms4-myc9 underwent a posttranslational modification concurrent with Cdc5 expression, as determined by SDS-PAGE (Figure 4A, lanes k and l). Because Cdc5 is involved in the control of several aspects of chromosome segregation and cells lacking meiotic expression of Cdc5 fail to progress beyond meiosis I (Clyne et al., 2003; Lee and Amon, 2003), it was necessary to uncouple defects in meiotic progression from the requirement for Cdc5 in the modification and activation of Mus81-Mms4. This was achieved by meiotic depletion of the APC/C activator Cdc20 (PCLB2-CDC20) (Lee and Amon, 2003), which led to the accumulation of cells in metaphase I but failed to affect Cdc5-dependent phosphorylation and hyperactivation of Mus81-Mms4 (Figure S4B, left panel). In contrast, cells lacking both Cdc20 and Cdc5 failed to modify and hyperactivate Mus81-Mms4, despite accumulating with meiosis I spindles (Figure S4B, right panel).

These data indicate that Cdc5 regulates the activity of Mus81-Mms4 during meiosis I, and that Cdc5-dependent phosphorylation of Mms4 could be important for the disengagement of DNA joint molecules. Recent work has shown that ectopic expression of Cdc5 in ndt80Δ cells (arrested in pachytene of prophase I) is sufficient to promote the resolution of HJs (Sourirajan and Lichten, 2008). We therefore determined whether expression of Cdc5 in ndt80Δ mutants is sufficient to regulate Mus81-Mms4, by generating MMS4-myc9 ndt80Δ mutants in which CDC5 expression was under the control of GAL4.ER (Sourirajan and Lichten, 2008). Addition of β-estradiol results in the specific induction of CDC5, while leaving the remaining genes in the NDT80 regulon off. Remarkably, induction of CDC5 was sufficient to promote the modification of Mms4 and prematurely boost the activity of Mus81-Mms4 during prophase I (Figure 4C, lanes d and e).

To confirm that the Cdc5-mediated hyperactivation of Mus81-Mms4 was a direct consequence of its phosphorylation, fully modified and hyperactive Mus81-Mms4 was prepared from MMS4-myc9 cells synchronized at metaphase I (PCLB2- CDC20). When treated with λ-phosphatase, but not inactivated phosphatase, we observed that increased electrophoretic mobility of Mms4 was linked with a reduction of Mus81-Mms4 nuclease activity (Figure 4D, lane c). Moreover, using cells expressing Myc-tagged Mms4, we found that Cdc5 was present in anti-Myc immunoprecipitates (Figure 4E, lanes h and i). These data demonstrate that Cdc5-dependent phosphorylation hyperactivates Mus81-Mms4 during meiosis I, and that Cdc5 promotes JM resolution during meiosis through the phosphorylation and activation of Mus81-Mms4.

Analysis of Phosphorylation-Defective Mutants of Mus81-Mms4

To identify the sites of phosphorylation on Mms4, we wished to immunoaffinity purify Mus81-Mms4 from large-scale cell cultures for mass spectrometry (MS). Parallel experiments, described later in this work, indicated that related Mus81-Mms4 phosphorylation events also take place in mitotic G2/M phase cells (Figure 6A and Figure S4C), allowing us to prepare large amounts of Mus81-Mms4 from synchronized mitotic cultures blocked at G2/M using the microtubule-depolymerizing drug benomyl.

Figure 6. Cell-Cycle Regulation of Mus81-Mms4 and Yen1 Activity in Mitotic Yeast.

Figure 6

Open in a new tab

(A) Cells expressing Mms4-myc9 were synchronized by a factor arrest/release, and extracts were analyzed by western blotting for the indicated proteins. Mms4-myc9 was immunoaffinity purified from extracts and assayed for HJ resolution activity. As, sample from asynchronous proliferating cells.

(B) As in (A), but using cells expressing Yen1-myc9 instead of Mms4-myc9.

(C) Inactivation of Mus81-Mms4 by dephosphorylation. Mms4-myc9 was immunoaffinity purified from G2/M cells collected 60 min after α factor release. Proteins were treated with λ-phosphatase as indicated and assayed for HJ resolution activity.

(D) Activation of Yen1 by dephosphorylation. Yen1-myc9 was immunoprecipitated from G2/M cells collected 25 min after α factor release. Proteins were analyzed as in (C).

See also Figure S7.

MS analysis of purified Mms4 led to the identification of 12 phosphorylated residues. Although interactions with Mus81 occur at the C-terminal region of Mms4 (Fu and Xiao, 2003), the phosphorylated residues were all located toward the N-terminal half of Mms4 (Figure 5B, detailed in Table S1). Further sites were indicated by sequence prediction, as the preferred binding sequence for the Polo-box domain is serine-phosphoserineproline (S-pS-P), overlapping with a CDK consensus sites (T/S-P-X-K/R, or the minimal consensus T/S-P) (Elia et al., 2003). The MMS4 sequence also contains five putative CDK-priming sites (Table S1), one of which (S56) has a perfect Cdc5-binding motif and was phosphorylated in vivo as determined by MS analysis. Due to incomplete peptide coverage, we were unable to confirm whether the other four predicted CDK sites were phosphorylated.

To determine the effects of Mms4 phosphorylation we generated an MMS4 allele, mms4-14A-ha3, in which 14 of the predicted/identified sites were mutated to alanine (Table S1). When Mus81-Mms4-14A was isolated from synchronized meiotic cultures, we observed basal levels of HJ resolvase activity that failed to be hyperactivated upon Cdc5 expression, consistent with reduced Cdc5-dependent phosphorylation (Figure 5A, lanes j–l). Additional control experiments confirmed that Mms4-14A was specifically defective in phosphorylation-dependent hyperactivation: (1) comparison of protein levels showed that Mms4-14A was expressed at similar levels to wild-type Mms4 (Figure 5A, Figure S4D, and Figure S5E); (2) immunoaffinity purification of Mus81-myc9 revealed normal associations with Mms4-14A (Figure 5A); (3) the nuclear localization of Mus81 throughout meiosis was normal in mms4-14A mutants (Figure S5A); (4) chromatin-associated nuclear foci that form during meiosis were present in cells expressing wild-type or phospho mutant Mms4-14A (Figure S5B); and (5) mms4-14A was able to partially suppress the DNA repair defects observed for mms4Δ mutants in response to DNA-damaging drugs such as methyl methanesulfonate (Figure S4E). Taken together, these data show that, in mms4-14A mutants, Mus81-Mms4 fails to become hyperactivated in meiosis I in response to Cdc5 accumulation and meiotic cell-cycle progression.

Joint Molecule Resolution and Chromosome Segregation Depend on Cdc5-Mediated Hyperactivation of Mus81-Mms4

Our data suggest a model in which Cdc5-mediated phosphorylation and hyperactivation of Mus81-Mms4 plays a key role in coordinating the timely completion of meiotic recombination with chromosome segregation. Consistent with this, cells expressing mms4-14A exhibited severe defects in chromosome segregation similar to those observed with mus81Δ (Figure 5C). We also found that the majority (82%) of mms4-14A yen1Δ double mutants failed to segregate their chromosomes during meiosis II, showing that this event is again largely dependent on the integrity of YEN1 (Figure 5C). Similarly, the efficiency of spore formation and viability of mms4-14A and mms4-14A yen1Δ double mutants was comparable to that observed with mms4Δ and mms4Δ yen1Δ mutants, respectively (Figure 5D and Figures S5C and S5D). Finally, despite being expressed at similar levels to Mms4-WT (Figure S5E), we found that Mms4-14A was unable to rescue the defect in mms4Δ yen1Δ mutants (Figure S5C).

To directly determine the effect of Cdc5-mediated hyperactivation of Mus81-Mms4 on JM resolution and CO formation, we monitored these events upon induction of Cdc5 expression in ndt80Δ cells carrying MMS4-WT, mms4Δ, or mms4-14A (Hunter and Kleckner, 2001; Sourirajan and Lichten, 2008). Physical analysis of recombination at the HIS4LEU2 locus showed that Cdc5 expression triggered the elimination of JMs and promoted the formation of CO recombinants in MMS4-WT but not mms4Δ cells (Figure 5E and Figure S6). Furthermore, mms4Δ mutants accumulated aberrant multichromatid JMs (mc-JMs). With mms4-14A cells, we observed a reproducible delay in the kinetics of JM elimination without any accumulation of mc-JMs (Figure 5E and Figure S6F). These results show that Cdc5-mediated activation of Mus81-Mms4 is required for the timely resolution of JMs, consistent with previous proposals (Sourirajan and Lichten, 2008), and also that Mus81-Mms4 operates independently of Cdc5 in the elimination of aberrant mc-JMs during prophase I.

Mitotic Regulation of Mus81-Mms4 and Yen1 Activity

To determine whether Mus81-Mms4 and Yen1 undergo analogous regulatory mechanisms during mitosis, anti-Myc immunoaffinity purifications of each protein were carried out using synchronized mitotic yeast carrying Mms4-myc9 or Yen1-myc9 after release from α factor arrest (G1/S). We found that both enzymes were tightly regulated throughout the mitotic cell cycle (Figures 6A and 6B). The nuclease activities of Mus81-Mms4 and Yen1 were low during S phase (15–30 min after release, see also FACS profile in Figure S7). As cells accumulated Cdc5 and the M phase cyclin Clb1, the activity of Mus81-Mms4 increased sharply (Figure 6A, 30–45 min). The peak of Mus81-Mms4 activity correlated with the peak of Cdc5 expression and the initiation of Clb1 proteolysis, which marks entry into anaphase I (60 min). A decline in activity was then observed as cells exited the first cell cycle and re-entered S phase (75–90 min). In the case of Yen1, a similar activity profile was observed, although the sharpness of the cycle was even greater than that of Mus81-Mms4 (Figure 6B). Activation of Yen1 occurred at a slightly later stage of mitosis, and was coordinated with cyclin degradation and entry into anaphase I (Figure 6B, 60 min). These results indicate that Mus81-Mms4 is activated in response to M phase entry, and that Yen1 is activated later as cells initiate anaphase.

Western blot analysis of the electrophoretic mobility of Mms4 and Yen1 revealed a striking correlation between activity levels and posttranslational modification (Figures 6A and 6B). In the case of Mus81-Mms4, nuclease activation correlated with increased Mms4 phosphorylation (Figure 6A). Again, we found that phosphatase treatment of Mus81-Mms4 isolated from cells at the peak of activation during M phase (60 min) resulted in increased electrophoretic mobility and inactivation of the nuclease activity (Figure 6C). In the case of Yen1, nuclease inactivation correlated with its phosphorylation, as seen by the slightly reduced electrophoretic mobility of Yen1 during S phase (Figure 6B). Importantly, λ-phosphatase treatment of inactive Yen1 isolated from S phase cells resulted in a dramatic increase in activity (Figure 6D). These data demonstrate that the activities of Mus81-Mms4 and Yen1 are tightly regulated throughout the cell cycle in proliferating cells, and that the mechanism of regulation appears similar to that seen during meiosis.

Regulation of MUS81-EME1 and GEN1 in Human Cells

Finally, we explored the regulatory control of the human orthologs of Mus81-Mms4 and Yen1. To do this, HeLa cells were generated that expressed MUS81 or GEN1 at endogenous levels from a bacterial artificial chromosome (BAC). Both proteins carried C-terminal FLAP tag fusions that allowed us to GFP-affinity purify the proteins. Immunoaffinity purified MUS81-EME1 and GEN1 were prepared from asynchronous (As) cells or from cultures blocked at various stages of the cell cycle using thymidine (Thy; G1/S phase arrest), camptothecin (CPT; S/G2 arrest), or nocodazole (NOC; prometaphase arrest). The proteins were then analyzed for their ability to cleave HJs in vitro, and again we found clear evidence for cell-cycle regulation. Whereas little MUS81-EME1 activity was seen in G1/S- or S/G2-arrested cells, a significant increase in HJ resolution activity was observed at prometaphase (Figure 7A, lane e).

Figure 7. Cell-Cycle Regulation of MUS81-EME1 and GEN1 Activity in Human Cells.

Figure 7

Open in a new tab

(A) Extracts were prepared from HeLa cells expressing MUS81-FLAP (CLJM6) after treatment with thymidine (Thy), camptothecin (CPT), or nocodazole (NOC), and analyzed by western blotting for the indicated proteins. MUS81-FLAP was affinity-purified from each sample and assayed for HJ resolution activity. Control samples were prepared from control asynchronous cells (As) or untransfected HeLa cells (U).

(B) As in (A), except that the HeLa cells expressed GEN1-FLAP (CLJM4). Extracts and affinity-purified GEN1-FLAP were assayed by western blotting and for HJ resolution activity.

(C) Immunofluorescence analysis of asynchronous HeLa cells expressing GEN1-FLAP. DNA was visualized by DAPI staining, and α-tubulin staining marks mitotic cells by decorating the mitotic spindle.

(D) Model for the timing and control of HJ resolution. In meiosis two waves of Holliday junction resolution ensure the elimination of recombination intermediates and promote the segregation of chromosomes at both meiotic divisions. Mus81-Mms4 activity is kept low until the onset of meiosis I when Cdc5 expression is induced. Cdc5 binds and phosphorylates Mms4, hyperactivating Mus81-Mms4. At this time, Yen1 is inactive, but becomes activated by dephosphorylation at meiosis II, where it acts as a safeguard that ensures chromosome segregation. In mitosis, the activities of Mus81-Mms4 and Yen1, and their human homologs MUS81-EME1 and GEN1, are enhanced as cells enter M phase of the cell cycle. Prior to this time, noncrossover-promoting pathways of junction dissolution are likely to be dominant. The regulation of the Mus81 and Yen1/GEN1 pathways occurs through cycles of phosphorylation/dephosphorylation similar to that of meiotic cells.

The role that the Polo-like kinase Cdc5 plays in the hyperactivation of Mus81-Mms4 in yeast prompted us to determine whether human Polo-like kinase PLK1 might be involved in the M phase activation of MUS81-EME1. We found that activation of MUS81-EME1 was coincident with reduced mobility of both MUS81 and EME1, and that PLK1 kinase was present in the MUS81-FLAP pull-downs from nocodazole-treated cells (Figure 7A, lane e). These results indicate that Polo kinase-mediated phosphorylation is likely to regulate the activity of MUS81-EME1, as observed in yeast.

In contrast to MUS81-EME1, we did not observe such tight regulation of GEN1 activity, although again maximal activity was observed at M phase (Figure 7B, lane e). However, analysis of the subcellular localization of GEN1 by immunofluorescence microscopy (visualization of the GFP-tag) revealed regulation by a second level of control (Figure 7C). In this case, GEN1 was predominantly found in the cytoplasm, except in cells that had a mitotic spindle (marked with α-tubulin), in which case it was distributed throughout the entire cell. These results suggest that GEN1 protein is controlled by two levels of regulation, first by cell-cycle-mediated changes to its activity, and second by preventing access of the nuclease to DNA until breakdown of the nuclear envelope.

DISCUSSION

In this work, we have uncovered a remarkable regulatory system that directs the outcome of joint molecule resolution, by timing the actions of crossover-promoting nucleases according to cellular needs. Moreover, we show that the completion and outcome of homologous recombination is precisely co-coordinated with cell-cycle progression, by mechanisms that ensure the specialized chromosome segregation programs of meiosis and mitosis. In meiotic cells, cell-cycle-regulated phosphorylation events control the enzymatic activities of Mus81-Mms4 and Yen1, which link the completion of recombination to CO generation and chromosome segregation. In mitotic cells, a similar regulatory network produces cycles of inactivation/activation that restrain the nuclease activities, biasing the engagement of JMs toward NCO-promoting pathways, and then releasing them to ensure that persistent intermediates are processed in a timely fashion for chromosome segregation.

Mus81-Mms4 Resolves Joint Molecules, whereas Yen1 Safeguards Chromosome Segregation

During meiosis, the processing of recombination intermediates by endonucleolytic resolution provides two essential functions. First, HJ resolvases sever the physical connections between chromosomes that would otherwise impede chromosome segregation. Second, the conversion of JMs to COs facilitates the bipolar segregation of homologous chromosomes. Therefore, segregation in general, and bipolar segregation in particular, are linked to the efficiency and outcome of JM resolution. These are known to be critical cellular events because resolution defects at meiosis I could contribute to the cosegregation of homologous chromosomes in human oocytes, which is directly associated with pregnancy loss and developmental disabilities (Hassold and Hunt, 2001).

Our study indicates that in yeast Mus81-Mms4 plays a leading role in the resolution of meiotic JMs and thereby promotes the timely segregation of homologs in meiosis I. mus81Δ and mms4Δ mutants undergo meiosis and generate spores, which are characterized by a small delay and reduction in CO formation, and low viability (de los Santos et al., 2001, 2003). Our analysis revealed that Mus81-Mms4 is required for JM resolution at meiosis I, and that in its absence a single round of chromosome segregation occurs at meiosis II. Importantly, Yen1 is essential for the single chromosome segregation event in mus81Δ mutants, despite being dispensable for meiosis in otherwise wild-type cells. Yen1 therefore provides a safeguard activity that deals with JMs that have escaped the attention of Mus81-Mms4. As a consequence, the phenotype of mus81Δ is modest in comparison with fission yeast that lacks Yen1 and relies almost entirely on Mus81-Eme1 for JM resolution and CO formation (Boddy et al., 2001; Osman et al., 2003).

Timing and Control of JM Resolution by Cell-Cycle-Regulated Phosphorylation

Chromosome segregation analyses revealed the existence of a degree of redundancy between Mus81-Mms4 and Yen1, but also highlighted a functional separation of the two JM resolution pathways in time. Consistent with our genetic data, we found that the activity of Mus81-Mms4 peaks at meiosis I, whereas Yen1 is activated to process HJs as cells undergo meiosis II (see model, Figure 7D). Previous work had established that dHJ accumulation/resolution is coordinated with cell-cycle progression through the actions of Cdc5 (Clyne et al., 2003; Sourirajan and Lichten, 2008). The present work extends this model, by demonstrating that Mus81-Mms4 is a direct target of Cdc5 in JM processing. Physical analysis of recombination showed that Mus81-Mms4 was required for Cdc5-mediated JM resolution. Furthermore, the analysis of mms4 mutants defective for Cdc5-mediated phosphorylation confirmed that modification of Mms4 was important for the efficient disengagement of JMs. These results explain the chromosome segregation defects observed in mms4-14A cells and underscore the importance of the precise coupling of JM resolution with cell-cycle progression. However, Mms4-14A was still capable of supporting JM resolution, albeit with a delay, raising the possibility that either Mms4-14A is still partially activated by Cdc5, or that phosphorylation of a second Cdc5 target may facilitate JM resolution in the presence of a basal level of Mus81-Mms4 activity. Cdc5 is known to play a role in SC disassembly (Sourirajan and Lichten, 2008) and components of the SC have been proposed to counteract Sgs1-mediated dHJ dissolution (Jessop et al., 2006; Oh et al., 2007; Rockmill et al., 2003). It is therefore possible that Cdc5-mediated SC disassembly may facilitate JM resolution by Mus81-Mms4.

In contrast to Mus81-Mms4, the phosphorylation of Yen1 keeps its activity under tight control by phosphorylation-dependent inhibition. Yen1 phosphoinhibition is initiated during premeiotic S phase and maintained until the onset of meiosis II, at which time dephosphorylation alleviates inhibition. Although the kinase responsible for Yen1 phosphorylation has yet to be identified, the phosphorylation profile observed would be compatible with several kinases involved in promoting S phase and controlling recombination (e.g., CDK, DDK). Previous studies proposed that Yen1 is a target of Clb5-CDK (Loog and Morgan, 2005), and its sequence encodes a multitude of predicted CDK consensus sites. Moreover, it has been shown that phosphorylated Yen1 is predominantly cytoplasmic in S phase, whereas in G1 or upon CDK downregulation it localizes to the nucleus (Kosugi et al., 2009). Most likely, phosphorylation-dependent subcellular relocalization provides a second level of regulatory control, similar to that observed with GEN1 in human mitotic cells.

Our studies indicate that in order to ensure that all JMs are resolved in time for chromosome segregation, the activities of Mus81-Mms4 and Yen1 are sequentially elevated. Therefore, if JMs escape the attention of Mus81-Mms4, a safeguard activity is in place. This back-up solution may have evolved to suppress the inability of cells to delay cell-cycle progression once JMs mature into dHJs, suggesting that Yen1 acts as a checkpoint substitute and that like most checkpoint proteins only becomes important when abnormal levels of stress are introduced into the system. To our knowledge, our study also provides the first demonstration of the direct regulation of the activity of a structure-specific nuclease. It is remarkable that the same type of posttranslational modification regulates the activity of both nucleases, but whereas phosphorylation activates Mus81-Mms4, it inhibits Yen1 activity. How posttranslational modification controls the biochemical activities of these nucleases remains to be determined.

Resolvase Regulation and Suppression of Crossover Formation in Mitosis

In contrast to meiosis, the processing of dHJs in mitosis is generally associated with formation of NCO products. In budding yeast, a complex of Sgs1-Top3-Rmi1 promotes dHJ dissolution reactions that exclusively form noncrossover products (Gangloff et al., 1994; Ira et al., 2003). Similar reactions occur in human cells, where BLM-TopoIIIα-RMI1-RMI2 (the BTR complex) combine to ensure that dHJs that link sister chromatids are dissolved into non-crossovers, as indicated by the elevated sister chromatic exchange (SCE) phenotype that is characteristic of Bloom’s Syndrome (BS) cells (Chaganti et al., 1974; Wu and Hickson, 2003). Recently, we showed that the high frequency of SCE formation in BS cells, which are mutated for BLM, could be reduced by downregulation of MUS81 and GEN1 (Wechsler et al., 2011). These results indicated that the BTR complex plays a primary role in dHJ processing, and that the high frequency of SCE formation results from the actions of MUS81 and GEN1, which resolve the JMs that persist in BS cells into either crossovers or non-crossovers.

The results described in this work provide insights into the interplay between HJ dissolution and resolution in mitotic cells. First, in yeast, we found that the activities of Mus81-Mms4 and Yen1 are low in S phase, at the time when JMs are formed, and that similar regulatory events occur in human cells. Thus, JM dissolution pathways are likely to have the upper hand in S phase cells, at least until Mus81-Mms4 (MUS81-EME1) is hyperactivated at G2/M, and Yen1 (GEN1) is activated and gains access to the DNA as cells enter mitosis (Figure 7D). One important prediction of this model is that the processing of a DSB generated at G2/M should have a lower bias toward NCO formation, as a consequence of activation of the nuclease pathways. Consistent with this prediction, increased CO formation is observed when DSBs are induced in nocodazole-treated cells (Ira et al., 2003). From these results, we propose that a primary function of Mus81 and Yen1 in dissolution-proficient cells is to capture and resolve any JM intermediates that have eluded the NCO-promoting pathways and persist until mitosis.

As detailed for meiotic and mitotic yeast, the MUS81-EME1 and GEN1 pathways are regulated by cycles of phosphorylation/dephosphorylation. Our results also indicate that PLK1 may play a key role in regulating MUS81-EME1 activity in a manner similar to that shown for Cdc5 in yeast. This is an interesting observation because PLK1 is overexpressed in many cancers and overexpression correlates with a poor prognosis and lower overall survival rate (Schöffski, 2009). One possibility is that the premature activation of MUS81-EME1 could result in an increased frequency of COs and loss of heterozygosity in these cancers.

EXPERIMENTAL PROCEDURES

All experimental procedures are described in detail in the Extended Experimental Procedures.

Yeast Strains and Cultures

All strains were derivatives of haploid BY4741 or diploid SK1, as detailed for each experiment in Table S2. Meiotic time courses and immunofluorescence microscopy of fixed cells were performed as described (Petronczki et al., 2006). Cells were grown for 11 hr (30°C), washed with sporulation medium (SPM, 2% potassium acetate) and inoculated into SPM to OD600 ~3.5. This time point was defined as t = 0 in all meiotic experiments. For synchronous release of mitotic cultures, BY4741 MATa derivatives were grown exponentially in YPD (OD600 ~0.3) at 30°C and synchronized by addition of α factor (final concentration 3 μM). After 2 hr (>95% unbudded cells), cells were harvested, washed once in YPD, and released in one half volume of YPD (t = 0 hr).

BAC-Mediated Protein Expression in HeLa Cells

Modified BACs containing GEN1-FLAP and MUS81-FLAP were transfected into HeLa cell lines and selected for stable integration and endogenous levels of expression (Poser et al., 2008).

Protein Assays

Meiotic and mitotic cellular lysates, and immunoaffinity-purified proteins were analyzed by SDS-PAGE through standard or phosphoaffinity gels. For nuclease assays, tagged proteins were immunoaffinity purified from yeast or human cell extracts using anti-Myc, or GFP-Trap (Chromotek) beads and washed extensively. The beads (approximate volume 10 μl) were then mixed with 10 μl cleavage buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, or 2.5 mM for Yen1) and ~1 nM 5′-32P-end-labeled synthetic Holliday junction X0 or X26 DNA (Ip et al., 2008). After 30 min incubation (or 1 hr for Mms4) at 30°C with gentle rotation, reactions were stopped by addition of 2.5 μl of 10 mg/ml proteinase K and 2% SDS, followed by incubation for 45 min at 37°C. Loading buffer (3 μl) was then added and radiolabeled products were separated by 10% native PAGE, dried on Whatman paper and analyzed by autoradiography and processed with ImageJ software, or by phosphorimaging using a Typhoon scanner and ImageQuant software. Resolution activity was calculated by determining the fraction of nicked duplex DNA product relative to the sum of the intact substrate and resolution product.

Dephosphorylation Assays

Myc-fusion proteins were affinity purified using anti-Myc agarose beads and washed extensively with buffer R (40 mM Tris, pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% NP40). The beads were then incubated with or without λ-phosphatase (NEB), or λ-phosphatase inactivated by heating to 95°C for 10 min. Protein samples were then washed extensively with cleavage buffer and analyzed by western blotting and in Holliday junction resolution assays.

Physical Analysis of Recombination at the HIS4LEU2 Locus

DNA physical assays were performed essentially as described (Hunter and Kleckner, 2001; Kim et al., 2010).

Supplementary Material

Supplementary Material

ACKNOWLEDGMENTS

We thank Wolfgang Zachariae and Nancy Kleckner for strains and plasmids, Tony Hyman for the BAC tagging cassettes, Keun Kim for help with the physical analysis of recombination, and members of our laboratory for comments and criticisms. This work was supported by Cancer Research UK, the European Research Council, the Louis-Jeantet Foundation, the Swiss Bridge Foundation, and the Breast Cancer Campaign. J.M. was a recipient of a Human Frontiers Science Program long-term Fellowship, and M.G.B. by the Angeles Alvariño program of the Xunta de Galicia (Spain).

Footnotes

SUPPLEMENTAL INFORMATION

Supplemental Information includes Extended Experimental Procedures, seven figures, and two tables and can be found with this article online at doi:10.1016/j.cell.2011.08.032.

REFERENCES

  1. Allers T, Lichten M. Differential timing and control of noncrossover and crossover recombination during meiosis. Cell. 2001;106:47–57. doi: 10.1016/s0092-8674(01)00416-0. [DOI] [PubMed] [Google Scholar]
  2. Blanco MG, Matos J, Rass U, Ip SCY, West SC. Functional overlap between the structure-specific nucleases Yen1 and Mus81-Mms4 for DNA-damage repair in S. cerevisiae. DNA Repair (Amst.) 2010;9:394–402. doi: 10.1016/j.dnarep.2009.12.017. [DOI] [PubMed] [Google Scholar]
  3. Boddy MN, Gaillard PHL, McDonald WH, Shanahan P, Yates JR, 3rd, Russell P. Mus81-Eme1 are essential components of a Holliday junction resolvase. Cell. 2001;107:537–548. doi: 10.1016/s0092-8674(01)00536-0. [DOI] [PubMed] [Google Scholar]
  4. Chaganti RS, Schonberg S, German J. A manyfold increase in sister chromatid exchanges in Bloom’s syndrome lymphocytes. Proc. Natl. Acad. Sci. USA. 1974;71:4508–4512. doi: 10.1073/pnas.71.11.4508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chu S, DeRisi J, Eisen M, Mulholland J, Botstein D, Brown PO, Herskowitz I. The transcriptional program of sporulation in budding yeast. Science. 1998;282:699–705. doi: 10.1126/science.282.5389.699. [DOI] [PubMed] [Google Scholar]
  6. Clyne RK, Katis VL, Jessop L, Benjamin KR, Herskowitz I, Lichten M, Nasmyth K. Polo-like kinase Cdc5 promotes chiasmata formation and cosegregation of sister centromeres at meiosis I. Nat. Cell Biol. 2003;5:480–485. doi: 10.1038/ncb977. [DOI] [PubMed] [Google Scholar]
  7. de los Santos T, Hunter N, Lee C, Larkin B, Loidl J, Hollingsworth NM. The Mus81/Mms4 endonuclease acts independently of double-Holliday junction resolution to promote a distinct subset of crossovers during meiosis in budding yeast. Genetics. 2003;164:81–94. doi: 10.1093/genetics/164.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. de los Santos T, Loidl J, Larkin B, Hollingsworth NM. A role for MMS4 in the processing of recombination intermediates during meiosis in Saccharomyces cerevisiae. Genetics. 2001;159:1511–1525. doi: 10.1093/genetics/159.4.1511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Elia AE, Rellos P, Haire LF, Chao JW, Ivins FJ, Hoepker K, Mohammad D, Cantley LC, Smerdon SJ, Yaffe MB. The molecular basis for phosphodependent substrate targeting and regulation of Plks by the Polo-box domain. Cell. 2003;115:83–95. doi: 10.1016/s0092-8674(03)00725-6. [DOI] [PubMed] [Google Scholar]
  10. Fricke WM, Brill SJ. Slx1-Slx4 is a second structure-specific endonuclease functionally redundant with Sgs1-Top3. Genes Dev. 2003;17:1768–1778. doi: 10.1101/gad.1105203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fu Y, Xiao W. Functional domains required for the Saccharomyces cerevisiae Mus81-Mms4 endonuclease complex formation and nuclear localization. DNA Repair (Amst.) 2003;2:1435–1447. doi: 10.1016/j.dnarep.2003.08.013. [DOI] [PubMed] [Google Scholar]
  12. Gangloff S, McDonald JP, Bendixen C, Arthur L, Rothstein R. The yeast type I topoisomerase Top3 interacts with Sgs1, a DNA helicase homolog: a potential eukaryotic reverse gyrase. Mol. Cell. Biol. 1994;14:8391–8398. doi: 10.1128/mcb.14.12.8391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Haber JE, Heyer WD. The fuss about Mus81. Cell. 2001;107:551–554. doi: 10.1016/s0092-8674(01)00593-1. [DOI] [PubMed] [Google Scholar]
  14. Hassold T, Hunt P. To err (meiotically) is human: the genesis of human aneuploidy. Nat. Rev. Genet. 2001;2:280–291. doi: 10.1038/35066065. [DOI] [PubMed] [Google Scholar]
  15. Ho CK, Mazón G, Lam AF, Symington LS. Mus81 and Yen1 promote reciprocal exchange during mitotic recombination to maintain genome integrity in budding yeast. Mol. Cell. 2010;40:988–1000. doi: 10.1016/j.molcel.2010.11.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hunter N, Kleckner N. The single-end invasion: an asymmetric intermediate at the double-strand break to double-Holliday junction transition of meiotic recombination. Cell. 2001;106:59–70. doi: 10.1016/s0092-8674(01)00430-5. [DOI] [PubMed] [Google Scholar]
  17. Ip SCY, Rass U, Blanco MG, Flynn HR, Skehel JM, West SC. Identification of Holliday junction resolvases from humans and yeast. Nature. 2008;456:357–361. doi: 10.1038/nature07470. [DOI] [PubMed] [Google Scholar]
  18. Ira G, Malkova A, Liberi G, Foiani M, Haber JE. Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast. Cell. 2003;115:401–411. doi: 10.1016/s0092-8674(03)00886-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jessop L, Rockmill B, Roeder GS, Lichten M. Meiotic chromosome synapsis-promoting proteins antagonize the anti-crossover activity of sgs1. PLoS Genet. 2006;2:e155. doi: 10.1371/journal.pgen.0020155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kaliraman V, Mullen JR, Fricke WM, Bastin-Shanower SA, Brill SJ. Functional overlap between Sgs1-Top3 and the Mms4-Mus81 endonuclease. Genes Dev. 2001;15:2730–2740. doi: 10.1101/gad.932201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Keeney S, Giroux CN, Kleckner N. Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family. Cell. 1997;88:375–384. doi: 10.1016/s0092-8674(00)81876-0. [DOI] [PubMed] [Google Scholar]
  22. Kim KP, Weiner BM, Zhang LR, Jordan A, Dekker J, Kleckner N. Sister cohesion and structural axis components mediate homolog bias of meiotic recombination. Cell. 2010;143:924–937. doi: 10.1016/j.cell.2010.11.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kosugi S, Hasebe M, Tomita M, Yanagawa H. Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs. Proc. Natl. Acad. Sci. USA. 2009;106:10171–10176. doi: 10.1073/pnas.0900604106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lee BH, Amon A. Role of Polo-like kinase CDC5 in programming meiosis I chromosome segregation. Science. 2003;300:482–486. doi: 10.1126/science.1081846. [DOI] [PubMed] [Google Scholar]
  25. Loog M, Morgan DO. Cyclin specificity in the phosphorylation of cyclin-dependent kinase substrates. Nature. 2005;434:104–108. doi: 10.1038/nature03329. [DOI] [PubMed] [Google Scholar]
  26. Mullen JR, Kaliraman V, Ibrahim SS, Brill SJ. Requirement for three novel protein complexes in the absence of the Sgs1 DNA helicase in Saccharomyces cerevisiae. Genetics. 2001;157:103–118. doi: 10.1093/genetics/157.1.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Niu H, Wan L, Baumgartner B, Schaefer D, Loidl J, Hollingsworth NM. Partner choice during meiosis is regulated by Hop1-promoted dimerization of Mek1. Mol. Biol. Cell. 2005;16:5804–5818. doi: 10.1091/mbc.E05-05-0465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Oh SD, Lao JP, Hwang PYH, Taylor AF, Smith GR, Hunter N. BLM ortholog, Sgs1, prevents aberrant crossing-over by suppressing formation of multichromatid joint molecules. Cell. 2007;130:259–272. doi: 10.1016/j.cell.2007.05.035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Oh SD, Lao JP, Taylor AF, Smith GR, Hunter N. RecQ helicase, Sgs1, and XPF family endonuclease, Mus81-Mms4, resolve aberrant joint molecules during meiotic recombination. Mol. Cell. 2008;31:324–336. doi: 10.1016/j.molcel.2008.07.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Osman F, Dixon J, Doe CL, Whitby MC. Generating crossovers by resolution of nicked Holliday junctions: a role for Mus81-Eme1 in meiosis. Mol. Cell. 2003;12:761–774. doi: 10.1016/s1097-2765(03)00343-5. [DOI] [PubMed] [Google Scholar]
  31. Petronczki M, Matos J, Mori S, Gregan J, Bogdanova A, Schwickart M, Mechtler K, Shirahige K, Zachariae W, Nasmyth K. Monopolar attachment of sister kinetochores at meiosis I requires casein kinase 1. Cell. 2006;126:1049–1064. doi: 10.1016/j.cell.2006.07.029. [DOI] [PubMed] [Google Scholar]
  32. Poser I, Sarov M, Hutchins JR, Hériché JK, Toyoda Y, Pozniakovsky A, Weigl D, Nitzsche A, Hegemann B, Bird AW, et al. BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals. Nat. Methods. 2008;5:409–415. doi: 10.1038/nmeth.1199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rass U, Compton SA, Matos J, Singleton MR, Ip SCY, Blanco MG, Griffith JD, West SC. Mechanism of Holliday junction resolution by the human GEN1 protein. Genes Dev. 2010;24:1559–1569. doi: 10.1101/gad.585310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rockmill B, Fung JC, Branda SS, Roeder GS. The Sgs1 helicase regulates chromosome synapsis and meiotic crossing over. Curr. Biol. 2003;13:1954–1962. doi: 10.1016/j.cub.2003.10.059. [DOI] [PubMed] [Google Scholar]
  35. Schöffski P. Polo-like kinase (PLK) inhibitors in preclinical and early clinical development in oncology. Oncologist. 2009;14:559–570. doi: 10.1634/theoncologist.2009-0010. [DOI] [PubMed] [Google Scholar]
  36. Schwacha A, Kleckner N. Identification of double Holliday junctions as intermediates in meiotic recombination. Cell. 1995;83:783–791. doi: 10.1016/0092-8674(95)90191-4. [DOI] [PubMed] [Google Scholar]
  37. Sourirajan A, Lichten M. Polo-like kinase Cdc5 drives exit from pachytene during budding yeast meiosis. Genes Dev. 2008;22:2627–2632. doi: 10.1101/gad.1711408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tay YD, Wu L. Overlapping roles for Yen1 and Mus81 in cellular Holliday junction processing. J. Biol. Chem. 2010;285:11427–11432. doi: 10.1074/jbc.M110.108399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wechsler T, Newman S, West SC. Aberrant chromosome morphology in human cells defective for Holliday junction resolution. Nature. 2011;471:642–646. doi: 10.1038/nature09790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wu L, Hickson ID. The Bloom’s syndrome helicase suppresses crossing over during homologous recombination. Nature. 2003;426:870–874. doi: 10.1038/nature02253. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material
NIHMS51351-supplement-01.pdf (1.3MB, pdf)

RESOURCES