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Mol Cell Biol. 2013 August; 33(16): 3365–3376.
PMCID: PMC3753904

Three Distinct Modes of Mec1/ATR and Tel1/ATM Activation Illustrate Differential Checkpoint Targeting during Budding Yeast Early Meiosis


Recombination and synapsis of homologous chromosomes are hallmarks of meiosis in many organisms. Meiotic recombination is initiated by Spo11-induced DNA double-strand breaks (DSBs), whereas chromosome synapsis is mediated by a tripartite structure named the synaptonemal complex (SC). Previously, we proposed that budding yeast SC is assembled via noncovalent interactions between the axial SC protein Red1, SUMO chains or conjugates, and the central SC protein Zip1. Incomplete synapsis and unrepaired DNA are monitored by Mec1/Tel1-dependent checkpoint responses that prevent exit from the pachytene stage. Here, our results distinguished three distinct modes of Mec1/Tec1 activation during early meiosis that led to phosphorylation of three targets, histone H2A at S129 (γH2A), Hop1, and Zip1, which are involved, respectively, in DNA replication, the interhomolog recombination and chromosome synapsis checkpoint, and destabilization of homology-independent centromere pairing. γH2A phosphorylation is Red1 independent and occurs prior to Spo11-induced DSBs. DSB- and Red1-dependent Hop1 phosphorylation is activated via interaction of the Red1-SUMO chain/conjugate ensemble with the Ddc1-Rad17-Mec3 (9-1-1) checkpoint complex and the Mre11-Rad50-Xrs2 complex. During SC assembly, Zip1 outcompetes 9-1-1 from the Red1-SUMO chain ensemble to attenuate Hop1 phosphorylation. In contrast, chromosome synapsis cannot attenuate DSB-dependent and Red1-independent Zip1 phosphorylation. These results reveal how DNA replication, DSB repair, and chromosome synapsis are differentially monitored by the meiotic checkpoint network.


Meiosis generates four haploid daughter cells from a diploid parental cell. The central steps of meiosis are the pairing and recombination of homologous chromosomes, followed by their segregation in two rounds of cell division. A key step of meiosis occurs in the pachytene stage, in which the homologous chromosomes (i.e., the parental chromosomes, each containing two sister chromatids) align (synapsis) (1, 2). Meiotic recombination is initiated by DNA double-strand breaks (DSBs) induced by Spo11, and chromosome synapsis is mediated by a tripartite structure named the synaptonemal complex (SC). The SC is a zipper-like protein complex that consists of a central element and two dense lateral/axial elements. The tripartite structure of the SC is strikingly conserved from budding yeast to humans, underscoring its prominent function during meiosis (24). In the budding yeast Saccharomyces cerevisiae, the central element of the SC includes a major component (Zip1) and the SC initiating proteins (Zip2-4, Mer3, Msh4-5, Spo16, and Ecm11-Gcm2) (515). The axial elements include the sister chromatid cohesin complex (Rec8/Scc3/Smc1/Smc3) and three meiosis-specific components, Hop1, Red1, and Mek1 (1, 16, 17). Red1 and Hop1, like Rec8, assemble along the rod-like cores of meiotic chromosomes (16). The SC proteins have different impacts on meiotic DNA recombination and cell cycle progression. In cell cycle progression, checkpoint mechanisms are signaling pathways that detect ongoing cell cycle events and relay this information to other (metabolically independent) processes. Checkpoint pathways, therefore, act as surveillance mechanisms that halt cell cycle progression and activate repair responses when necessary. When SC assembly or repair of Spo11-induced DNA DSBs is incomplete, a checkpoint mechanism is activated that prevents exit from the pachytene stage of meiotic prophase and meiotic nuclear division in cells that fail to complete meiotic recombination and synapsis. This checkpoint is variously referred to in the literature as the “pachytene checkpoint,” the “chromosome synapsis checkpoint,” or the “meiotic recombination checkpoint” (1, 18, 19). Some reports have suggested that the meiotic recombination checkpoint mainly monitors the leptotene-zygotene transition, when recombination is committing to crossovers and the SC is initiating, not the pachytene stage (2, 6), hinting at the elaborate nature of the meiotic checkpoint network and our thus-far inadequate understanding of the process. It has further been reported that meiotic DNA repair and chromosome synapsis are monitored independently in budding yeast (20) and in Caenorhabditis elegans (21).

In budding yeast, the meiotic checkpoint network requires Mec1 and Tel1, the yeast homologs of mammalian DNA damage sensor kinases ATR and ATM (2224). These two kinases participate in the DNA damage checkpoint during mitosis and meiosis: Tel1 and its binding partner Tel2 are recruited by the Mre11-Rad50-Xrs2 (MRX) complex to unresected DSBs (2528), and Mec1 is recruited to replication protein A (RPA)-coated single-stranded (ssDNA) tails via its binding partner Ddc2 (29). Ddc2 localizes to meiotic chromosomes and accumulates in yeast mutants (such as hop2Δ and dmc1Δ) that accumulate unrepaired ssDNA. The formation of meiotic Ddc2 foci is defective in the absence of wild-type RPA (30). Mec1 activation also requires three additional DNA damage sensors: the yeast 9-1-1 complex (Ddc1-Mec3-Rad17), its clamp loader Rad24-RFC complex, and Dpb11 (2224, 29).

Mec1 and Tel1 preferentially phosphorylate their substrates at serine (S) and threonine (T) residues that precede glutamine residues, so-called SQ/TQ motifs. Many known targets of the ATM/ATR family proteins contain SQ/TQ cluster domains (SCDs). For example, Mec1 and Tel1 phosphorylate Hop1 at multiple SQ/TQ motifs within its SCD (amino acid residues 298 to 329), among which the phosphorylation of T318 affects Hop1 activities (e.g., SC assembly and interhomolog recombination) the most profoundly. Hop1-T318 phosphorylation is further critical for chromosomal recruitment and activation of Mek1 (31, 32), a meiosis-specific protein kinase that upholds interhomolog bias (3338). Mec1 and Tel1 also phosphorylate SQ and TQ sites in several other phosphoproteins during meiosis, including histone H2A at S129 (H2A-S129 [γH2A]), Rad53, RPA, Sae2, and Zip1 (3943). The Mec1/Tel1-dependent phosphorylation of Zip1 (32, 42) dynamically destabilizes homology-independent centromere pairing in response to DSBs (42). Protein phosphatase 4 (PP4) is responsible for the dephosphorylation of several Mec1/Tel1-dependent phosphoproteins (e.g., Hop1, Zip1, and H2A-S129) during meiosis (42).

Considerable evidence also indicates that Smt3, the small ubiquitin-like modifier (SUMO) protein in budding yeast, plays key roles in mediating SC assembly and in activating the meiotic checkpoint processes (4447). First, Red1, Zip1, and Zip3 can all physically interact with Smt3, poly-Smt3 chains (SUMO chain), and Smt3 conjugates (collectively referred to as SUMO-C) via their Smt3-interacting motifs (44, 46, 47). Second, the results of two-hybrid assays and genetic analyses suggest that Red1 and Zip1 might sandwich the SUMO chain to mediate SC assembly (47). This model is supported by recent evidence that sumoylation at Lys153 of the SUMO E2 conjugating enzyme Ubc9 controls SUMO chain formation and chromosome synapsis in meiosis (48). Moreover, Zip3 and other ZMM proteins might promote SC elongation by ensuring that Zip1 orients perpendicularly to the longitudinal axis of the SC (44, 47). Third, the Red1-SUMO chain interaction is required for activating Mec1 and Tel1 to phosphorylate Hop1 (47). Also of note, it has been reported that Red1 could bind and activate the 9-1-1 checkpoint complex to promote H2A-S129 phosphorylation (49).

In this work, we determined the functional relationship between the Red1-SUMO chain interaction and the Red1–9-1-1 interaction and their roles in activating Mec1 and Tel1 for phosphorylation of three different targets (H2A-S129, Zip1-S75, and Hop1-T318). Unexpectedly, our results revealed that Mec1 and Tel1 are activated differentially to phosphorylate these targets during yeast meiotic prophase. We also showed that chromosome synapsis specifically attenuates Hop1-T318 phosphorylation but not Zip1-S75 phosphorylation or H2A-S129 phosphorylation at late sporulation time points. We suggest, therefore, that one of the functions of the SC is to repress persistent Hop1 phosphorylation.


Yeast strains, two-hybrid assay, and physical analysis.

All meiotic experiments were performed using diploid cells in isogenic SK1 strains. Quantitative yeast two-hybrid assays, tetrad dissection, and physical analysis were carried out as previously described (32, 44, 47).

Antisera and quantitative Western blot analyses.

Antisera against Hop1, phosphorylated Hop1-T318, Zip1, phosphorylated Zip1-S75, and phosphorylated Rad54-T132 were as recently described (32). Anti-phospho-H2A-S129 antiserum (49) was from Millipore, Billerica, MA, USA. Quantitative Western blot analyses were performed as recently described and repeated 2 to 4 times (32).


Two isoleucine residues (I758 and I761) of Red1 are critical for the Red1-SUMO interaction.

Red1, the main axial/lateral element protein of the SC, plays key roles in coupling three central events of meiosis: interhomolog recombination, chromosome synapsis, and meiotic checkpoint signaling. These meiotic functions of Red1 depend on its interactions with SUMO chains or conjugates (i.e., SUMO-C) (47) and the 9-1-1 complex (49). The Smt3 interacting motif (residues 758 to 761 [ISII]) of Red1 (Fig. 1A) is required for the Red1–SUMO-C interaction, and the SUMO-C binding mutant (red1 with I758 mutated to R [red1I758R]) is defective in activating Mec1 and Tel1 for Hop1 phosphorylation and spore viability (47). Previously, we reported that wild-type Red1C (residues 611 to 827) displayed strong interactions with Smt3 and that the Red1CI758R mutant was defective in this interaction (47). Here, we further showed that the Red1CI760,761R double mutant also exhibited much weaker interactions with Smt3. In contrast, the Red1CI760R mutant was still able to bind to Smt3 (Table 1). These results indicate that I758 and I761 are functionally critical for binding to SUMO or SUMO-C.

Fig 1
Red1–SUMO-C interaction is required for activation of Mec1 and Tel1 for Hop1-T318 phosphorylation but not for Zip1-S75 or H2A-S129 phosphorylation. (A) Schematic representation of full-length Red1, showing the SUMO interaction motif, the Mec3 ...
Table 1
Two-hybrid analyses

Red1-Ddc1 mutant can associate with SUMO chains or conjugates.

The C terminus of Red1 also contains two interaction sites for the 9-1-1 complex: the Mec3 interaction site is located between residues 531 and 551, and the Ddc1 interaction site resides between residues 729 and 751 of Red1 (Fig. 1A). Previously, it was reported that the production of viable spores was only moderately affected in the red1-Mec3 (red1Q537A,V540A) mutant. In contrast, the red1-Ddc1 (red1I743A) and red1-Ddc1-Mec3 (red1I743A,Q537A,V540A) mutants were defective in spore viability and meiotic checkpoint signaling (i.e., H2A-S129 phosphorylation). Red1-Mec3, Red1-Ddc1, and Red1-Ddc1-Mec3 were defective in interaction with Mec3, Ddc1, and both, respectively (49).

Although the Smt3 interacting motif and the 9-1-1 interaction sites are located close to one another in Red1, their functional relationship is still unclear. We found that the Red1C-Ddc1(I743A) mutant displayed moderate two-hybrid interaction with Smt3. The order of affinity of these proteins for Smt3 was Red1C > Red1CI760R > Red1C-Ddc1 > Red1CI760,761R > Red1CI758R (Table 1). To further verify the affinity of Red1 variant proteins for Smt3 chains, we expressed the full-length Red1 fusion proteins glutathione S-transferase (GST)-Red1-His6, GST-Red1I758R-His6, and GST-Red1-Ddc1-His6 in Escherichia coli and then sequentially purified them on Ni2+ and glutathione resins. SUMO chains were synthesized in vitro and partially purified by size exclusion gel filtration (see Fig. S1A in the supplemental material) as described previously (47). GST-His6 was used as a negative control. The order of affinity of these proteins for SUMO chains was GST-Red1-His6 > GST-Red1-Ddc1-His6 [dbl greater-than sign] GST-Red1I758R-His6 ~ GST-His6 (see Fig. S1B). Thus, unlike Red1I758R, Red1-Ddc1 could associate with SUMO chains. Additional biochemical, genetic and cytological studies further revealed that red1-Ddc1 is a hypomorphic allele and Red1-Ddc1 can partly activate both Mec1 and Tel1 (see below; see also Fig. S2 in the supplemental material).

Red1–SUMO-C interaction is a prerequisite for the Red1–9-1-1 interaction.

To further reveal the relationship between the Red1–SUMO-C interaction and the Red1-Ddc1 interaction, we also examined whether Ddc1 might interact with the four Red1C mutants (Red1CI758R, Red1C-Ddc1, Red1CI760R, and Red1CI760,761R). Our results revealed that Red1C-Ddc1, Red1CI760,761R, and Red1CI758R were all defective in binding to Ddc1. In contrast, Red1CI760R displayed reduced interactions with Ddc1 (Table 1). Because the order of affinity of these proteins for Smt3 was Red1C > Red1CI760R > Red1C-Ddc1 > Red1CI760,761R > Red1CI758R (Table 1), we inferred that the Red1-Smt3 interaction is necessary for the Red1-Ddc1 interaction.

Additional two-hybrid analyses further revealed that both Red1C and Smt3 exhibited weak two-hybrid interactions with Mec3 and Rad17, respectively. We also found that neither Red1C nor Smt3 exhibited interaction with Rad24 (Table 1). Rad24 is a component of the clamp loader Rad24-RFC complex that is required for Mec1-dependent checkpoint function in meiosis (22, 31). Because the Smt3 interacting motif and the 9-1-1 interaction sites are located close to one another in Red1, the Red1–SUMO-C interaction likely functions in bridging the Red1–9-1-1 interaction. However, because all three components of the 9-1-1 complex display weak or no two-hybrid interactions with Smt3, we further inferred that a SUMO-C dependent conformation change in Red1 might increase the Red1–9-1-1 interaction. According to this hypothesis, the Red1–9-1-1 interaction will become much weaker when the SUMO chains and/or conjugates (i.e., SUMO-C) are absent or greatly reduced in vivo.

Previously, we also reported that Red1 preferentially interacted with the Smt3 chain but not the Smt3 monomer. Red1C and Smt3 induced a high level of two-hybrid interaction in smt3-allR reporter cells. In contrast, Red1C and Smt3-allR exhibited a much weaker interaction in the same reporter cells (47). The smt3-allR allele encodes a mutant protein in which the nine lysine residues in Smt3 are replaced with arginines. The Smt3-allR mutant is capable of SUMO conjugation to target proteins (including Smt3) but produces many fewer SUMO chains (50). We further demonstrated that Ddc1 and Red1C displayed strong two-hybrid interactions in SMT3 reporter cells. In contrast, when the same two-hybrid assay was carried out in smt3-allR reporter cells, Ddc1 hardly associated with Red1C (Table 1). These results support the model that the Red1-SUMO chain (or Red1–SUMO-C) interaction is a requirement for the Red1-Ddc1 interaction.

Red1–SUMO-C interaction affects Red1 meiotic phenotypes.

To further reveal the functional relationship between the Red1–9-1-1 interaction and Red1–SUMO-C interactions in vivo, we next compared their effects on spore viability and Hop1-T318 phosphorylation using yeast strains expressing wild-type or mutant V5 epitope-tagged Red1 (V5-Red1). Hop1-T318 phosphorylation is critical for spore viability and interhomolog recombination (31, 32). The order of spore viability was V5-RED1 (95%) > V5-red1I760R (65%) > V5-red1-Ddc1 (20%) > V5-red1-Ddc1-Mec3 (15%) > V5-red1I760,761R (2%) > V5-red1I758R (<1%) ~ red1Δ (<1%) (see Table S1 in the supplemental material). All yeast strains were then induced to undergo relatively synchronous sporulation. At the time points indicated in the figures (Fig. 1B to toD),D), cells were harvested for preparation of total cell lysates according to the TCA precipitation protocol described previously (44, 47). Hop1 phosphorylation was monitored by utilizing phosphorylation-induced shifts in electrophoretic mobility (46, 47) or by an antibody specific to phosphorylated Hop1-T318 (32). Consistent with the results of the two-hybrid interaction and spore viability assays, we found that the order of phosphorylated Hop1-T318 levels was V5-RED1 > V5-red1-Ddc1 [dbl greater-than sign] V5-red1I758R ~ red1Δ (Fig. 1B) and V5-RED1 ~ V5-red1I760R > V5-red1-Ddc1-Mec3 > V5-red1I760,761R (Fig. 1C). Unlike the V5-red1I758R and red1Δ mutants, the V5-red1I760,761R mutant displayed reduced Hop1-T318 phosphorylation (Fig. 1C) and generated 2% viable spores (see Table S1). Together, our results confirm that the Red1-SUMO chain interaction is critical for the meiotic function of Red1 in promoting Hop1-T318 phosphorylation, interhomolog recombination, and spore viability.

Red1–SUMO-C interaction mediates both Mec1- and Tel1-mediated Hop1-T318 phosphorylation.

Mec1 and Tel1 kinases contribute to the phosphorylation of Hop1 and Hop1-T318 in wild-type cells and most mutant genotypes (31, 32). In contrast, Hop1 phosphorylation is reportedly mediated by Tel1 but not Mec1 in rad50S cells (31). The rad50S mutation blocks the resection of the DSB ends (51). Since the red1Δ or red1I758R mutants exhibit no detectable Hop1 or Hop1-T318 phosphorylation, the Red1-SUMO chain ensemble might also act with the MRX complex to activate Tel1 for Hop1 phosphorylation. To examine this possibility, we introduced the rad50S mutation into the V5-RED1, V5-red1I758R, V5-red1-Ddc1, and red1Δ strains. Western blot time course analyses showed that significant portions of Hop1 proteins were phosphorylated in V5-RED1 rad50S and V5-red-Ddc1 rad50S strains. In contrast, Hop1 was not phosphorylated in the V5-red1I758R rad50S or red1Δ rad50S mutant (Fig. 1D). Thus, Tel1-mediated Hop1 phosphorylation is completely dependent on the Red1–SUMO-C ensemble. Recently, it was reported that budding yeast MRX is a positive regulator of DNA damage-induced sumoylation during vegetative growth and that Xrs2 and Mre11 are SUMO modified in response to DSBs (52). Here, we further showed by two-hybrid assays that Mre11 (but not Xrs2) exhibited strong interactions with Smt3. Mre11 further exhibited weak two-hybrid interactions with Red1C, and the Mre11-Red1C interaction was stronger than the Mre11-RedCI758R interaction (Table 1). These results indicate that the Red1–SUMO-C ensemble might transiently associate with the MRX complex and activate Tel1 for Hop1 phosphorylation.

Red1–SUMO-C interaction is not required for Mec1- and Tel1-mediated Zip1-S75 phosphorylation and H2A-S129 phosphorylation.

Next, we examined whether Zip1 phosphorylation and H2A-S129 phosphorylation also depend on the Red1–SUMO-C interaction. Zip1 phosphorylation was monitored with anti-phospho-Zip1-S75 antibodies or anti-Zip1 antibodies by utilizing a phosphorylation-induced shift in electrophoretic mobility. H2A-S129 phosphorylation was monitored with anti-H2A-S129 antibodies and anti-H2A antibodies, respectively (32). Our data indicated that neither H2A-S129 phosphorylation nor Zip1-S75 phosphorylation was significantly affected by red1Δ, V5-red1-Ddc1, V5-red1-Mec3-Ddc1, and V5-red1I758R mutations in the wild-type RAD50 background (Fig. 1B and andC)C) or in the rad50S background (Fig. 1D). We therefore concluded that the Red1, the Red1–SUMO-C, and the Red1-Ddc1 interactions are not essential for Zip1-S75 phosphorylation and H2A-S129 phosphorylation.

Contrary to our results here, it was previously reported that the red1-Ddc1 mutant is defective in H2A-S129 phosphorylation (49). This discrepancy is apparently due to the use of yeast strains with dmc1Δ mutations in the previous study (49): compared to the red1-Ddc1 dmc1Δ mutant, the RED1 dmc1Δ mutant only displayed H2A-S129 hyperphosphorylation at the 24-h time point. Because the RED1 dmc1Δ mutant had already been arrested in the late stages of prophase by 8 to 10 h after the initiation of meiosis (49, 53), the H2A-S129 hyperphosphorylation observed at 24 h might not result from a primary defect in prophase chromosome metabolism.

Genetic requirements of Mec1/Tel1 activation for differential target phosphorylation.

Because Red1 or the Red1–SUMO-C interaction is required for Hop1-T318 phosphorylation but not H2A-S129 phosphorylation or Zip1-S75 phosphorylation (Fig. 1), we further inferred that Mec1 and Tel1 might be differentially activated to phosphorylate different substrates in response to Spo11-generated DSBs. To probe this possibility, we compared the phosphorylation of four different protein targets (H2A-S129, Zip1-S75, Hop1-T318, and Rad54-T132) in the V5-RED1, V5-RED1 spo11Δ, and red1Δ strains (Fig. 2A). Phosphorylated Hop1-T318 is required for chromosome recruitment and activation of Mek1 (31, 32), which then phosphorylates Rad54 at T132 (36). We found that Hop1-T318 phosphorylation and Rad54-T132 phosphorylation required both Spo11 and Red1. In contrast, H2A-S129 phosphorylation still occurred in the spo11Δ mutant and the red1Δ mutant. Zip1 phosphorylation in the same cultures was also monitored as described above (Fig. 1). The upper Zip1 bands and the phosphorylated Zip1-S75 bands appeared in the wild type and the red1Δ mutant but not in the spo11Δ mutant (Fig. 2A). These results further confirmed that Zip1 and Zip1-S75 phosphorylation requires Spo11-generated DSBs (42) but is independent of Red1 (see below). In contrast, H2A-S129 phosphorylation occurs independently of Spo11 and Red1.

Fig 2
Genetic requirements for phosphorylation of different Mec1/Tel1-dependent targets. (A) Western blot time course analysis in V5-RED red1Δ and V5-RED spo11Δ backgrounds. (B) Western blot time course analysis in rad51Δ dmc1Δ ...

Unrepaired DSBs are insufficient to trigger Hop1 phosphorylation in the absence of Red1.

Red1 (16, 54) and the Red1–SUMO-C interaction (47) are required for promoting wild-type levels of Spo11-generated DSBs, and DSBs are more rapidly repaired via intersister recombination in the absence of Red1 or its downstream effector Mek1 kinase (33, 34). DSBs can also be repaired via the synthesis-dependent strand-annealing pathway to yield noncrossover products (55, 56). The absence of Hop1 or Hop1-T318 phosphorylation in the red1Δ or V5-red1I758R strain might simply result from faster DSB repair via intersister recombination or the synthesis-dependent strand-annealing pathway, respectively. This hypothesis was examined here by comparing Hop1 and Hop1-T318 phosphorylation in rad51Δ dmc1Δ, red1Δ rad51Δ dmc1Δ, and zip1Δ rad51Δ dmc1Δ mutants. The rad51Δ dmc1Δ double mutant persistently accumulated resected DSBs (or ssDNA) and produced markedly fewer crossover products than the rad51Δ or dmc1Δ single mutant (57). Contrary to the hypothesis, quantitative Western blot analysis showed that Hop1-T318 phosphorylation was detected in rad51Δ dmc1Δ and zip1Δ rad51Δ dmc1Δ mutants but not in the red1Δ rad51Δ dmc1Δ mutant (Fig. 3B and andC).C). As shown in Figure 1D, Red1 and the Red1–SUMO-C interaction are also essential for Hop1 phosphorylation in the rad50S mutant that accumulated unprocessed DSB ends. Taken together, we conclude that neither unprocessed DSB ends nor unrepaired ssDNA alone is sufficient to promote Hop1 or Hop1-T318 phosphorylation in the absence of Red1.

Fig 3
Mec1/Tel1-dependent phosphorylation of three targets in sporulating wild-type, ndt80Δ, zip3Δ, and zip3Δ ndt80Δ strains. (A) Western blot time course analysis of phosphorylation of different targets in NDT80 and ndt80Δ ...

Previously, it was reported that the Mec1/Ddc2 complex is recruited to RPA-coated ssDNA tails during yeast meiotic prophase: Ddc2 localizes to meiotic chromosomes and accumulates in yeast mutants (such as hop2Δ and dmc1Δ strains) that accumulate unrepaired ssDNA, and the formation of meiotic Ddc2 foci is defective in an RPA mutant (30). Moreover, the Xrs2 subunit of the MRX complex could recruit Tel1 to unprocessed DSB ends and then activate Tel1 (25, 26, 28). Therefore, based on our data shown in Figures 1C and and2,2, neither of these two pathways is sufficient to activate Tel11 and Mec1 for Hop1 phosphorylation in the absence of Red1. Instead, the Red1–SUMO-C ensemble is required for MRX and 9-1-1 to activate Tel1 and Mec1 for Hop1 phosphorylation in response to unprocessed DSB ends and unrepaired ssDNA, respectively.

We also found that Zip1 did not affect Hop1-T318 phosphorylation in the rad51Δ dmc1Δ double mutant (Fig. 2B and andC),C), consistent with the report that the Dmc1- and Rad51-mediated strand exchange reactions precede SC assembly (6). Finally, phosphorylated Zip1 and Zip1-S75 were detected in rad51Δ dmc1Δ and red1Δ rad51Δ dmc1Δ mutants but not in the zip1Δ rad51Δ dmc1Δ mutant. Because the rad50S strain and the rad51Δ dmc1Δ double mutant persistently accumulate unprocessed DSB ends and unrepaired ssDNA, respectively, the MRX-Tel1 pathway (26, 27) and the ssDNA-RPA-Ddc2/Mec1 pathway (30) can mediate Zip1-S75 phosphorylation (Fig. 1D and and2B)2B) regardless of whether Red1 is present or not.

Hop1-T318 phosphorylation and Zip1-S75 phosphorylation are differentially regulated in the ndt80Δ mutant that arrests at the pachytene stage of meiosis.

Next, we investigated whether chromosome synapsis affects Mec1/Tel1-dependent target phosphorylation (Fig. 3A). Ndt80 is a meiosis-specific transcription factor that activates the expression of more than 200 genes at midmeiosis, including those required for meiotic divisions and spore formation (58). The ndt80Δ mutant fails to exit pachytene, arresting with persistent full-length SCs and partly repaired DSBs but unresolved joint molecules (5961).

The results of quantitative Western blot analyses (Fig. 3A and andB)B) revealed that both Hop1-T318 phosphorylation and Zip1-S75 phosphorylation diminished greatly after the 7-h time point in the wild-type cells (Fig. 3A, left). In contrast, only Zip1-S75 phosphorylation but not Hop1-T318 phosphorylation was persistently accumulated in ndt80Δ cells (Fig. 3A, center right). Both strains displayed similar H2A-S129 phosphorylation. Next, the meiotic DSB levels of the same sporulation cultures were also analyzed by pulsed-field gel electrophoresis (Fig. 3C) and Southern analysis using an FDC1 DNA probe (Fig. 3D). The FDC1 gene (YDR539W) is located ~18 kbp from the right-arm telomere of chromosome IV. DSBs appeared in both strains at the 3-h time point. Most wild-type cells had completed DNA repair at the 7-h time point, as indicated by the reemergence of full-length linear chromosome IV species by this point (Fig. 3D, left). In contrast, nearly all ndt80Δ chromosome IV remained in the gel, in agreement with the presence of unresolved joint molecules (Fig. 3D, center right). Together, these results indicate that chromosome synapsis specifically attenuates Hop1-T318 phosphorylation but not Zip1-S75 phosphorylation or H2A-S129 phosphorylation.

The Red1–SUMO-C assembly can interact with 9-1-1 and MRX to activate Mec1 and Tel1 for Hop1-T318 phosphorylation (Table 1 and Fig. 1 and and2).2). Previously, using a yeast two-hybrid assay, we also found that the C-terminal portion of Zip1 protein (Zip1C; amino acid residues 849 to 975) and Red1 sandwich the SUMO chains to mediate SC assembly (47). Here, we further demonstrated that the Red1C-Ddc1 two-hybrid interactions were significantly reduced by the wild-type Zip1C protein but not the Zip13L-3R mutant (Table 1). The Zip13L-3R mutant, constructed by mutating three leucines (856 to 858) into three arginines, is defective in two-hybrid interactions with Smt3 (44). Therefore, Zip1, as a SUMO chain binding protein, can likely outcompete 9-1-1 (and/or MRX) for the Red1-SUMO chain ensemble during SC assembly. These results are consistent with the notion that Zip1-mediated chromosome synapsis apparently has a unique role in attenuating Hop1-T318 phosphorylation. Unfortunately, we were unable to coimmunoprecipitate Zip1 or the 9-1-1 complex with V5-Red1 from meiotic cell lysates. We reported previously (46, 47) that vacuolar proteases (e.g., Pep4, Prb1, and Prc1) are extremely troublesome for studying the biochemistry of yeast meiosis because these proteases are not only highly induced upon starvation but are also essential for sporulation. The Zip1 proteins (47) and the 9-1-1 components (data not shown) became extremely unstable after the meiotic cells were lysed in native buffers containing a variety of protease inhibitors.

Deletion of ZMM genes leads to persistent Hop1 phosphorylation.

Because the ndt80Δ mutant arrests at the pachytene stage with persistent full-length SCs, we suggest that one of the functions of the SC is to repress persistent Hop1 phosphorylation. To further verify this hypothesis, we also compared the three Mec1/Tel1-dependent target phosphorylations side-by-side in zip3Δ (Fig. 3A, center left) and zip3Δ ndt80Δ (Fig. 3A, right) mutants. Zip3 is a meiosis-specific SUMO E3 ligase required for the initiation of SC assembly (44). The zip3Δ mutant forms very few SCs but accumulates large aggregates of SC-related materials (i.e., polycomplexes) (44, 45). Our results indicated that the zip3Δ mutation resulted in persistent Hop1-T318 phosphorylation and Zip1-S75 phosphorylation, and both resulting levels of phosphorylation were much higher in the ndt80Δ mutant than in the NDT80 strain after the 6-h time point (Fig. 3B). Thus, Zip3 has a specific role in attenuating Hop1-T318 phosphorylation in strains at late sporulation time points.

The results of pulsed-field gel electrophoresis and Southern analysis using an FDC1 DNA probe (Fig. 3C and andD)D) further revealed that most zip3Δ cells completed DNA repair after the 12-h time point, as indicated by the reemergence of full-length linear chromosome IV species (Fig. 3D, center left). The zip3Δ ndt80Δ strain chromosomes IV, as in the ndt80Δ mutant, accumulated unresolved joint molecules (Fig. 3D, right). Notably, deletion of the ZIP3 gene in both strains also resulted in unrepaired and/or late-occurring DSBs. These DSBs, together with the unresolved joint molecules, contributed further to persistent Zip1-S75 and Hop1-T318 hyperphosphorylation in the zip3Δ and zip3Δ ndt80Δ mutants.

The zmm mutants, like the zip3Δ mutant, are defective in chromosome synapsis. Next, we applied quantitative Western analyses to compare Hop1-T318 phosphorylation and/or Zip1-S75 phosphorylation in several other zmm mutants in the ndt80Δ (see Fig. S3 in the supplemental material) and NDT80 (see Fig. S4 in the supplemental material) backgrounds. We found that all of the zmm ndt80Δ double mutants and the zmm single mutants examined here displayed persistent Hop1-T318 phosphorylation at later sporulation time points than the ndt80Δ mutant or the wild-type NDT80 strain.

Differential roles of the Sgs1 helicase in attenuating Hop1-T318 phosphorylation in zip1Δ and zip3Δ strains.

ZMM proteins are known to antagonize the anticrossover activities of Sgs1, which disassembles nascent crossover-designated recombination intermediates. The sgs1Δ null mutant and the sgs1ΔC795 mutant have limited effects on crossover recombination in wild-type cells (6264). The sgs1ΔC795 allele encodes only the first 652 amino acids of the protein and is lacking the helicase domain (65, 66). The sgs1ΔC795 mutant behaves like the sgs1Δ null mutant in several meiotic phenotypes: a much greater increase in crossovers was seen when either the sgs1Δ or sgs1ΔC795 mutation was introduced into zmmΔ mutants than was seen in the wild-type strain. They displayed increases in the numbers of axial associations between homologous chromosome cores (i.e., pseudosynapsis) in a zip1Δ background (64). In other zmmΔ null mutants, Sgs1 also prevents close juxtaposition of the axes of homologous chromosomes; for example, sgs1Δ zip3Δ and sgs1ΔC795 mer3Δ mutants restored full homolog axial association (by Red1 staining) to more than half of the nuclei. Of the chromosome pairs in which axes were closely juxtaposed, most displayed end-to-end Zip1 staining (i.e., truly synapsed) (62).

We showed by quantitative Western blot analyses that the order of the levels of phosphorylated Hop1-T318 for the various mutations was sgs1ΔC795 zip1Δ > zip3Δ ~ zip1Δ > zip3Δ sgs1ΔC795 > sgs1Δ zip3Δ ~ sgs1Δ C795 ~ wild type (Fig. 4). Both sgs1ΔC795 and sgs1Δ can attenuate Hop1-T318 phosphorylation in the zip3Δ strain. The sgs1Δ mutation has a stronger effect in attenuating Hop1-T318 phosphorylation in the zip3Δ strain, consistent with a previous report that the zip3Δ sgs1Δ double mutant cells could form full SC (50%) and partial SC (40%) (62). Contrary to results for the sgs1ΔC795 zip3Δ and sgs1Δ zip3Δ double mutants, we found that the sgs1Δ C795 zip1Δ double mutant exhibited higher levels of phosphorylated Hop1-T318 than the zip3Δ or zip1Δ mutant. These results indicate that Zip1-mediated SC assembly (in zip3Δ sgs1ΔC795 and sgs1Δ zip3Δ strains) but not Zip1-independent pseudosynapsis or interhomolog recombination (in the zip1Δ sgs1ΔC795 strain) is responsible for attenuating Hop1-T318 phosphorylation. These results further support the model that Zip1-mediated chromosome synapsis has a unique role in attenuating Hop1-T318 phosphorylation. We inferred that Sgs1 might have a role in preventing supercomplex formation between Zip1 and the Red1–SUMO-C ensemble (see Discussion).

Fig 4
Zip1-mediated chromosome synapsis attenuates Hop1-T318 phosphorylation in the absence of the wild-type SGS1 gene. (A) Western blot time course analysis of wild-type, sgs1ΔC795, zip1Δ, zip3Δ, zip1Δ sgs1ΔC795, zip3 ...

H2A-S129 phosphorylation appears at the onset of premeiotic DNA replication.

Almost all the strains examined here displayed persistent H2A-S129 phosphorylation, indicating that Spo11, Rad51, Dmc1, Red1, Zip1, and Ndt80 are not essential for H2A-S129 phosphorylation (Fig. 1 to to3).3). Next, we examined whether the onset of DNA replication induced H2A-S129 phosphorylation in meiosis. Two B-type cyclins, Clb5 and Clb6, activate Cdc28 kinase to initiate premeiotic DNA replication (6769) and Spo11-induced DSBs (7073). Using a method previously described to inhibit cdc28-as1 using 1-NM-PP1 {1-(1,1-dimethylethyl)-3-(1-naphthalenylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine} (67), we performed “execution point” analyses in V5-RED1 cdc28-as1 (46) to determine the timing of H2A-S129 phosphorylation during meiosis. The cell-permeable 1-NM-PP1 is an inhibitor of kinases that have been mutated by a single base substitution to become “analog sensitive (as)” compared to the wild-type kinase (74). Treatment of cdc28-as1 diploid cells with 5 μM 1-NM-PP1 (Fig. 5A) blocked meiotic DNA replication so that the cellular DNA content remained at 2N for more than 8.5 h (67). In the absence of 1-NM-PP1, almost all cdc28-as1 diploid cells accumulated 4N DNA content by 5 h. This arrest was caused by specific inhibition of Cdc28-as1 kinase, because DNA replication was blocked only by the combination of the cdc28-as1 mutation and 5 μM 1-NM-PP1 but not by either condition alone (67). Here, cdc28-as1 diploid cells were first induced to undergo relatively synchronous sporulation. At the indicated meiotic time points (0 to 8 h), 1-NM-PP1 (5 μM) was added to 5-ml aliquots of sporulation medium, and then the cultures were allowed to undergo sporulation. The cells were all harvested at 8 h for Western blot analysis. As expected, the addition of 1-NM-PP1 at the time points between 0 h and 3 h prevented DNA replication (Fig. 5A) until 8 h. In contrast, DNA replication (Fig. 5A) and H2A-S129 phosphorylation (Fig. 5B) began to appear if 1-NM-PP1 was added after the 3.5-h time point, and H2A-S129 phosphorylation reached maximum steady-state levels after the 4.5-h time point. On the other hand, Hop1-T318 phosphorylation began to appear between the 4.5-h and 5-h time points (Fig. 5B). Thus, H2A-S129 phosphorylation appeared at the onset of premeiotic S phase and then was slightly enhanced in response to Spo11-induced DSBs. Our results are consistent with previous reports that, during vegetative growth and in the absence of exogenous genotoxins, H2A-S129 phosphorylation in budding yeast (75) and fission yeast (76) occurred during S-phase and marked specific chromosomal domains that trigger DNA damage responses.

Fig 5
H2A-S129 phosphorylation appears at the onset of premeiotic DNA replication. (A) Progression of premeiotic DNA replication. Execution point analysis was carried out by adding 5 μM 1-NM-PP1 at the indicated time points and harvesting the sporulating ...


The present study demonstrated that Mec1 and Tel1 kinases are activated by three distinguishable pathways during early meiosis and that each pathway preferentially phosphorylates the SQ/TQ motif(s) of different protein targets (Fig. 6). (i) DSB- and Red1-independent H2A-S129 (γ-H2A) phosphorylation is mediated by Mec1 and Tel1 at the onset of meiotic DNA replication. (ii) Zip1-S75 phosphorylation (42) is DSB dependent but Red1 independent. Unprocessed DSBs and unrepaired ssDNA might recruit the MRX complex and the RPA-Ddc2/Mec1 ensemble to activate Tel1 (this study) and Mec1 (30) for Zip1 phosphorylation, respectively. (iii) Hop1-T318 phosphorylation occurs in a DSB- and Red1-dependent manner. Unlike Zip1-S75 phosphorylation, unprocessed DSBs and unrepaired DNA are insufficient to promote Hop1 phosphorylation in the absence of Red1 or if Red1 is unable to noncovalently interact with SUMO chains or SUMO conjugates. We suggest that the poly-SUMO chain or SUMO conjugates play a central role in coupling Spo11-induced DSBs, chromosome axes (via Red1), and interhomolog recombination and checkpoint activation (via Hop1 phosphorylation). The hypothetical model presented in Figure 6 explains why Hop1 phosphorylation but not Zip1 phosphorylation requires Red1. Red1 and Hop1 are structural components of chromosome axes, and these two proteins are loaded onto chromosome axes prior to SC assembly (or Zip1). In response to Spo11-induced DSBs, the Red1–SUMO-C ensemble recruits the MRX-Tel1 ensemble and 9-1-1/Mec1 to phosphorylate the neighboring Hop1 proteins. During SC assembly, Zip1 binds to the Red1–SUMO-C ensemble and outcompetes the 9-1-1 complex (or MRX). Mec1 (or Tel1) is also excluded from the SC; therefore, the SC components (e.g., Zip1 and Hop1) will no longer be phosphorylated by Mec1 (or Tel1). Meanwhile, PP4 dephosphorylates the phosphoylated Hop1 proteins (32). Further biochemical and structural studies will be necessary to reveal the coupling mechanisms between Spo11-DSBs, Red1, poly-SUMO chains or conjugates, the 9-1-1 complex, and the MRX complex.

Fig 6
The DNA replication checkpoint, DNA damage checkpoint, and chromosome synapsis checkpoint are independently monitored during yeast meiosis. Our results distinguished three modes of Mec1/Tec1 action in yeast meiosis that led to phosphorylation of three ...

A potential implication of our findings is that differentially targeted phosphorylation by Mec1 and Tel1 results from distinct DNA lesions. First, phosphorylated H2A-S129 forms in response to Spo11-independent DSBs during meiotic S phase, including natural replication fork barriers or chromosome fragile sites (77). Next, there might be substantial differences between Spo11-induced DSBs. For example, DSBs near or accessible to the chromosome axes (78) might have a better chance of functionally integrating with Red1 for Hop1 phosphorylation, thus promoting interhomolog recombination and SC formation. In contrast, DSB ends and their resulting ssDNA tails that are distant from the chromosome axes or unreachable by Red1 (i.e., in a red1Δ strain), may activate Tel1 and Mec1 only for Zip1-S75 phosphorylation but not for Hop1-T318 phosphorylation. Accordingly, they are likely to be repaired by homologous recombination using the sister chromatid as the template, which is not productive for homolog segregation during the first meiotic nuclear division.

The ndt80Δ mutant exhibited persistent Zip1-S75 phosphorylation and H2A-S129 phosphorylation but not Hop1-T318 phosphorylation (Fig. 4A and andB).B). This finding suggests that chromosome synapsis specifically attenuates Hop1-T318 phosphorylation, as the ndt80Δ mutant arrests at the pachytene stage with persistent full-length SCs. Since the ndt80Δ mutant accumulates unresolved joint molecules or partly repaired DNA (5961), it will be of interest to further determine whether these recombination intermediates are responsible for the persistent Zip1-S75 phosphorylation in the ndt80Δ mutant.

Our results shown in Figure 4 suggest that Sgs1 helicase activity prevents the formation of a complex between Zip1 and the Red1-SUMO chain ensemble. Sgs1 and Srs2 have overlapping roles in suppressing crossovers during double-strand break repair in yeast (79). Srs2 helicases and their E. coli homolog UvrD helicase both display antirecombinase activity to displace Rad51 and RecA recombination protein filaments, respectively, from ssDNA (80, 81). Further investigation will be required to determine whether and how Sgs1 functions to prevent protein-protein interactions between Zip1, Red1, and the SUMO chains (or SUMO conjugates). In fact, Sgs1 is known to be functionally relevant to the SUMO chains in vivo. Sgs1 is essential for yeast strains lacking the Slx5-Slx8 SUMO-targeted ubiquitin ligase (82). Deletion of the ULP2 SUMO isopeptidase gene or a ulp2-D623H hypomorphic allele suppresses sgs1Δ slx5Δ synthetic lethality and the slx5Δ sporulation defect (83). Intriguingly, Ulp2 has a function in promoting the accumulation of SUMO chainsin vivo (50). The ulp2-D623H slx5Δ sgs1Δ triple mutant strain displayed even higher levels of sumoylated proteins (or poly-SUMO chains) than a corresponding slx5Δ ulp2-D623H double mutant (83). Accordingly, Sgs1 can either promote the degradation of SUMO chains (e.g., via another yeast SUMO isopeptidase Wss1 [84]) or simply prevent the extension of SUMO chains. Based on our findings shown in Figure 4, we speculate that Sgs1 might achieve these functions by disrupting protein-protein interactions between SUMO chains and the SUMO-binding proteins or the Ubc9 E2 ligase (for extension of poly-SUMO chains), respectively.

Finally, a recent genome-wide study (85) strongly implicated budding yeast SUMO chains in replication-associated DNA damage and, also, in the maintenance of the normal higher order of chromatin structure during vegetative growth, including chromosome compaction, telomere clustering, nucleolar DNA organization, and spindle functions. The molecular details of this phenomenon are not yet understood. As reported in this study, poly-SUMO chains and/or conjugates apparently have a bridging function in coupling the axial element component Red1 to the 9-1-1 complex and the MRX complex for Mec1/Tel1 activation and to Zip1 for meiotic synapsis, respectively. From our observations, we further infer that the capability of poly-SUMO chains in associating with more than two SUMO binding proteins may account for their multiple roles in chromatin regulation. Additional exploration of SUMO chain-containing protein complexes and their dynamic regulation may shed further light on the biological functions of the SUMO system.

Supplementary Material

Supplemental material:


This work was supported by Academia Sinica and the National Science Council, Taiwan.

We thank Douglas Bishop (The University of Chicago, Chicago, IL, USA) for useful suggestions in manuscript writing and Miranda Loney (Agricultural Biotechnology Research Center, Academia Sinica) for English editing. We thank Andreas Hochwagen for sharing his unpublished results.

We declare that we have no conflict of interest.



While we were preparing our manuscript, Andreas Hochwagen and colleagues (New York University, New York, NY) also independently discovered that only Zip1 phosphorylation but not Hop1 phosphorylation was persistently accumulated in ndt80 cells.


Published ahead of print 17 June 2013

Supplemental material for this article may be found at


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