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S. cerevisiae Mre11 recruits conjugated SUMO moieties to facilitate the assembly and function of the Mre11-Rad50-Xrs2 complex

Double-strand breaks (DSBs) in chromosomes are the most challenging type of DNA damage. The yeast and mammalian Mre11-Rad50-Xrs2/Nbs1 (MRX/N)-Sae2/Ctp1 complex catalyzes the resection of DSBs induced by secondary structures, chemical adducts or covalently-attached proteins. MRX/N also initiates two parallel DNA damage responses—checkpoint phosphorylation and global SUMOylation—to boost a cell's ability to repair DSBs. However, the molecular mechanism of this SUMO-mediated response is not completely known. In this study, we report that Saccharomyces cerevisiae Mre11 can non-covalently recruit the conjugated SUMO moieties, particularly the poly-SUMO chain. Mre11 has two evolutionarily-conserved SUMO-interacting motifs, Mre11SIM1 and Mre11SIM2, which reside on the outermost surface of Mre11. Mre11SIM1 is indispensable for MRX assembly. Mre11SIM2 non-covalently links MRX with the SUMO enzymes (E2/Ubc9 and E3/Siz2) to promote global SUMOylation of DNA repair proteins. Mre11SIM2 acts independently of checkpoint phosphorylation. During meiosis, the mre11SIM2 mutant, as for mre11S, rad50S and sae2Δ, allows initiation but not processing of Spo11-induced DSBs. Using MRX and DSB repair as a model, our work reveals a general principle in which the conjugated SUMO moieties non-covalently facilitate the assembly and functions of multi-subunit protein complexes.


INTRODUCTION
Small ubiquitin-like modifier (SUMO) is a small regulatory protein found in almost all eukaryotic organisms (1)(2)(3). In the yeast Saccharomyces cerevisiae, the essential gene SMT3 encodes the SUMO protein. SUMOylation is a posttranslational modification, in which SUMO is covalently attached to a substrate protein including SUMO itself, to form a poly-SUMO chain. SUMOylation is mediated by an enzymatic cascade that is analogous to the one that is involved in ubiquitination. The removal of the SUMO adduct from targets is catalyzed by specific SUMO proteases, e.g. Ulp1 and Ulp2. The conjugated SUMO moieties (CSMs) are recognized by two types of SUMO-binding motifs, short hydrophobic sequences known as SUMO-interacting motifs (SIMs) (4) and the ZZ zinc fingers (5,6). The addition, removal and recognition of SUMO are influenced by and affect a plethora of cellular pathways. Because SUMOylation frequently targets entire groups of physically interacting proteins rather than individual proteins, it has been proposed that protein-group SUMOylation functions to establish new physical interactions between proteins that have SUMO-binding motifs (7). Alternatively, CSMs can covalently or non-covalently prevent premature aggregation by increasing the water solubility of individual protein subunits (8,9) prior to their assembly into a functional protein complex (10)(11)(12). A proof-of-concept has been provided by simultaneously expressing three capsid proteins of the footand-mouth disease virus (FMDV); these three SUMO fusion proteins formed a stable heterotrimeric complex. The proteolytic removal of SUMO moieties from the ternary complexes resulted in virus-like particles with a size and shape resembling the authentic FMDV, which contains 20 heterotrimers of the capsid proteins (10).
SUMOylation is strongly connected to the repair of DNA double-strand breaks (DSBs) (13). In S. cerevisiae vege-tative cells, mutations or deletion of SUMOylation genes cause a pronounced sensitivity to DNA damage and genomic instability, including the poly-SUMO chain mutant (smt3-allR) (14), E2 conjugating enzyme (ubc9), E3 ligase enzymes (siz1 , siz2 , mms21) (7,15), deSUMOylation proteases (ulp1 ts , ulp2 ) (16,17) as well as SUMO-targeted ubiquitin ligases (STUbLs; slx5 , slx8 ) (18,19). In parallel, with checkpoint phosphorylation, vegetative yeast cells also induce SUMOylation of many proteins that are needed for replication and repair in response to DNA damage (7,15). The MRX (Mre11-Rad50-Xrs2)-Sae2/Com1 ds-DNA endonuclease complex has been implicated as a positive regulator for DSB-induced global SUMOylation (15). Mre11 exhibits notable two-hybrid interactions with Ubc9 and Siz2, and it has been proposed that the binding of Siz2 might be achieved through Ubc9-catalyzed SUMOylation of Mre11 (7). Sae2 (also called Com1) (20) is a SUMOylated protein during vegetative growth, and SUMOylation of Sae2 increases both soluble Sae2 and the MRX function in DNA end resection (21). Sae2 apparently mediates removal of the MRX complex from the DNA damaged sites during vegetative growth. The MRX complex is retained at DSB ends in sae2 , thus turning on the DNA damage checkpoint to stall cell cycle progression (22,23). Recently, it has been reported that, following the resection of DSBs, Siz2 also collaborates with the ssDNA binding complex RPA (replication protein A) to enhance global SUMOylation (7,24). The relationship between MRX and RPA in promoting DSB-induced SUMOylation and the molecular mechanism of the interaction between MRX and Sae2 interactions remains unclear.
MRX has multiple functions during S. cerevisiae meiosis (25,26). First, it has a unique role in Spo11-induced DSBs independently of its catalytic activity, and the C-terminal portion of Mre11 is specifically required for this function (27,28). Second, the MRX-Sae2 endonuclease complex acts at each 5 -end of DSBs to generate 3 -end ss-DNA tails through the removal of a covalently linked Spo11-oligonucleotide complex (29,30). The 3 -end ssDNA tails subsequently assemble into nucleoprotein filaments comprised of two RecA-family recombinases (Rad51 and Dmc1) and their accessory factors to catalyze DSB repair via homologous recombination (31)(32)(33). Third, MRX senses DSBs and activates the Tel1 ATM checkpoint kinase for target phosphorylation. This checkpoint phosphorylation has dual roles in preventing superfluous Spo11-induced DSBs (34,35) and in promoting interhomolog recombination (12,35,36). Interhomolog recombination is a hallmark of meiotic recombination. A few Spo11-induced DSBs must be repaired using a homologous non-sister chromosome (but not a sister chromatin) as template to generate new combinations of DNA sequences (26).
Accumulating evidence has also revealed that SUMOylation functionally links two groups of S. cerevisiae proteins that are essential for interhomolog recombination. The first group includes three meiosis-specific chromosomal proteins Hop1, Red1 and Mek1. These proteins are the axial components of the synaptonemal complex (SC)--a zipper-like proteinaceous structure that mediates chromosome synapsis between homologous chromosomes during meiotic prophase. The SC consists of two dense lateral/axial elements and a central element. To assemble the SC, both the SC central protein, Zip1, and the SC axial protein, Red1, non-covalently interact with conjugated SUMO moieties (CSMs), such as poly-SUMO chains or conjugates. During SC assembly, the SC initiation protein, Zip3, acts as a SUMO E3 ligase that promotes the formation of additional CSMs (11,37). Consistent with these findings, it has been shown that the SUMOylation of Ubc9 promotes the formation of a poly-SUMO chain, which is a key event for SC formation (38). Furthermore, SUMOylation and the ubiquitin-mediated removal of CSMs (e.g. SUMOylated topoisomerase II or Red1) have been implicated in SC-mediated crossover interference (39). Crossover interference is a genetic phenomenon in which crossovers tend to be evenly spaced along any given meiotic chromosomes. SUMOylation is also critical in the regulation of meiotic recombination or chromosomal morphogenesis in other sexually-reproductive organisms, including the fission yeast S. pombe (40), the fungus Sordaria (41), Arabidopsis thaliana (42) and mammals (43).
Here, we investigate the molecular mechanism and physiological impacts of the Mre11-SUMO interaction in response to DSBs during vegetative growth and meiosis. Our results reveal that the yeast S. cerevisiae Mre11 can noncovalently recruit CSMs to facilitate both global SUMOylation and DSB repair.

Yeast strains, two-hybrid assay and physical analysis
All vegetative experiments were performed using haploid cells from isogenic W303 strains as described previously (15,56). All meiotic experiments were performed using diploid cells from isogenic SK1 strains. Quantitative yeast two-hybrid assays, tetrad dissection, fluorescence-activated cell sorting (FACS), pulsed-field gel electrophoresis (PFGE) and Southern hybridization were carried out as previously described (11,36,37).
To prepare the total cell lysates, the cell suspension was mixed with 1/2 volume of 0.5-mm acid-washed glass beads (Sigma), vigorously vortexed (30 s of vortexing and 30 s on ice) five times, and then microcentrifuged (16 000 x g) at 4 • C for 10 min. The supernatants were collected and incubated with IgG Sepharose beads (Sigma) or immobilized anti-HA affinity resin (Sigma) at 4 • C for 2 h. The precipitants were washed three times with 1 ml of lysis buffer and then resuspended in 100 l Laemmli loading buffer containing 100 mM dithiothreitol. The proteins were analyzed by SDS-PAGE and immunoblotting as described previously (11).
These two distinct possibilities were further examined by two-hybrid assays with either vegetative or meiotic twohybrid reporter cells (11,59). We found that Mre11 preferentially interacts with CSMs rather than the Smt3 monomer; the hierarchy for two-hybrid interactions with Mre11 was Smt3 > Smt3-allR > Smt3-GG ∼ = mock control in both vegetative and meiotic reporter cells (Table 1). Smt3-allR cannot form a polymeric chain because the nine lysine residues in the wild type Smt3 are replaced by arginine, but it remains competent in the SUMO conjugation with all target proteins (including wild-type Smt3) (60). Smt3-GG is a conjugation-incompetent Smt3 mutant that lacks the C-terminal pair of glycines required for E1-mediated Smt3 activation (61). Next, we constructed two SIM mutant proteins (Mre11 I9R and Mre11 I158R ), each with a mutation from isoleucine (I) to arginine (R). We found that neither of these two mutants exhibited notable two-hybrid interactions with Smt3, Smt3-allR or Smt3-GG (Table 1).
S. cerevisiae Mre11 exhibits strong two-hybrid interactions with the SUMO E2 enzyme Ubc9 and the SUMO E3 ligase Siz2 (7), and Mre11 is also a SUMOylated protein (7,11). We examined whether the Mre11-Ubc9 and Mre11-Siz2 interactions are mediated via SUMOylated Mre11 or non-covalently via the Mre11-CSM ensembles. We found that the hierarchy for the two-hybrid interaction with Ubc9 or Siz2 348-726 was Mre11 > Mre11 I158R ∼ = Mre11 I9R ∼ = mock control (Table I). Siz2 348 -726 is the C-terminal domain of Siz2 and harbors two SIMs (11). These results suggest that both interactions are likely mediated via the Mre11-CSM ensembles rather than SUMOylated Mre11 (also see below).
To minimize the potential effects of arginine replacement on protein structure or folding, we also constructed additional Mre11 SIM1 and Mre11 SIM2 mutants with one, two or three alanine mutations, respectively. All these mutants exhibited reduced or no two-hybrid interactions with Smt3, Smt3-allR or Smt3-GG (Supplementary file, Supplementary Table S1). The hierarchy for the twohybrid interaction of the Mre11 SIM1 mutants with Smt3

Mre11 I9R is defective in MRX assembly
Mre11 displays strong two-hybrid interactions with Xrs2 and Rad50 (62). Using both vegetative and meiotic twohybrid reporter cells (11,59), we found that Mre11 I158R (as for Mre11) exhibited notable two-hybrid interactions with Xrs2 and Rad50. In contrast, Mre11 I9R failed to interact with Rad50 and Xrs2 in the same assay (Table 1).
Next, we constructed three W303 strains (MRE11-6HA, mre11 I9R -6HA, mre11 I158R -6HA) that express an HA-epitope-tagged wild-type and mutant Mre11, respectively. Compared to MRE11-6HA and mre11 I158R -6HA, the mre11 I9R -6HA mutant displayed a slow vegetative growth phenotype on the YPD plate ( Figure 1B). The hierarchy for resistance to the DNA damage agent methyl Figure  1B). Again, to minimize the potential effects of arginine replacements on protein structure or folding, we also expressed and compared different Mre11 SIM1 -6HA and Mre11 SIM2 -6HA mutants for MMS resistance in a SK1 mre11 mutant ( Figure 1C). For all the mutants examined here, their two-hybrid interactions with Smt3 correlated well with the hierarchy for MMS resistance ( Figure 1C): The S. cerevisiae MRX is also required for telomere length maintenance. Like mre11 , mre11 D16A exhibits severe phenotypes in DNA repair and telomere maintenance. Mre11 D16A is defective in MRX assembly and lacks endonuclease activity (56). We first confirmed that Mre11 D16A exhibits defects in two-hybrid interactions with Smt3 and Rad50 but not Xrs2 (Table 1). Next, we showed that the hierarchy for telomere length maintenance was MRE11 ≥ Figure 1D). Genomic DNA was isolated from all strains, digested with XhoI and Southern hybridized for telomeric sequences. Consistent with previous reports, the telomeres in mre11 are shorter than those in mre11 D16A (56) and the sae2 deletion does not affect the maintenance of normal telomere length (63). These results indicate that mre11 I9R is phenotypically similar to mre11 , confirming that Mre11 I9R is defective in MRX assembly. In contrast, mre11 I158R -6HA showed much milder defects in MMS-resistance ( Figure 1B,C) and telomere maintenance ( Figure 1D). Thus, Mre11 I158R apparently can form a functional (at least partly) MRX complex.
D16 is one of seven essential and evolutionarilyconserved amino acid residues that coordinate two Mn 2+ ions to form the phosphodiesterase active site (64). Because Mre11 D16A is defective in association with the Mn 2+ , A16 and its neighboring amino acid residues (such as SIM1: IRIL, residues 9-12) might be improperly folded. As a result, Mre11 D16A is unable to interact with SUMO. Alternatively, D16 should be classified as being part of SIM1, or the SIM1-CSM interaction might mask D16 from chelating with Mn 2+ . In this scenario, Mre11 first recruits CSMs to facilitate Mre11 folding and/or MRX assembly. After replacement of CSMs with Mn 2+ , the folded Mre11 protein or the assembled MRX complex is no longer able to associate with CSMs via SIM1. This possibility is more logical, because it is consistent with the results of our two-hybrid (Table 1) and pull-down experiments (see below) showing neither Mre11 I9R nor Mre11 I158R is unable to interact with CSMs.

Mre11 I9R and Mre11 I158R are differentially defective in their ability to repair spontaneous and MMS-induced DNA damage
When DNA replication forks are stalled by spontaneous or MMS-induced DNA lesions, checkpoint proteins are activated to stabilize those stalled forks. Stalled forks eventually collapse and produce broken DSBs. The recovery of DNA replication is typically controlled by MRX-dependent repair of DSBs (65)(66)(67). Here, we compared the recovery of DNA replication in MRE11-6HA, mre11 I9R -6HA and mre11 I158R -6HA (Figure 2A). Cells were first arrested in G1 by mating pheromone ␣-factor and then released into S phase; this time point was referred to as T0. After 10 min, different amounts of MMS (0, 0.01%, 0.033%) were added to the cells for 45 min. This time point was referred to as T55, i.e. 55 min after release from ␣-factor arrest. Next, MMS was removed from the cells for 3 h (referred to as T235) to determine the recovery of DNA replication (Figure 2A). DNA replication was monitored by fluorescenceactivated cell sorting (FACS; Figure 2B) and pulsed-field gel electrophoresis (PFGE; Figure 2C). Checkpoint activation was determined by Western blot analysis with anti-Rad53 antisera ( Figure 2D). Rad53 is an essential DNA damage checkpoint protein kinase that is required for cell-cycle arrest in response to DNA damage. Rad53 activation occurs through direct phosphorylation by Mec1 ATR and Tel1 ATM followed by Rad53 autophosphorylation (68,69). The hyperphosphorylated Rad53 protein is distinguishable from the unphosphorylated Rad53 protein, as the latter migrates faster in a SDS-PAGE gel (68).
The steady-state levels of these three HA-tagged Mre11 proteins were not significantly different at T0, T55 or T235 ( Figure 2D). To examine their protein stability, we performed cycloheximide shut-off experiments (Supplementary Figure S1A). Protein synthesis was inhibited by 200 g/ml of cycloheximide added into the vegetative cultures. Samples were taken at 0, 30, 60, 90, 120 and 180 min after the addition of cycloheximide (Supplementary Figure  S1A, lower panel). The immunoblotting results revealed that Mre11-6HA, Mre11 I9R -6HA and Mre11 I158R -6HA exhibited similar half-lives (t 1/2 ≈45-60 min) in the presence of cycloheximide up to 180 min (Supplementary Figure  S1C), suggesting that these two SIM mutations have little or no effect on Mre11-6HA protein stability during vegetative growth.
Our results also indicate that, in the absence of MMS, the DNA damage checkpoint was activated at T55 and T235 in mre11 I9R -6HA but not in MRE11-6HA or mre11 I158R -6HA, as a proportion of the Rad53 proteins had become hyperphosphorylated and migrated more slowly in an SDS-PAGE gel ( Figure 2D, middle panel). In the presence of 0.01% MMS, the hierarchy for the DNA damage checkpoint responses was mre11 I9R -6HA > mre11 I158R -6HA > MRE11-6HA. In contrast, in the presence of 0.033% MMS, the DNA damage checkpoint responses in all three strains were distinctly activated at T55. The activated checkpoint in MRE11-6HA was then attenuated at T235, as the majority of the Rad53 proteins became unphosphorylated. Hyperphosphorylated Rad53 was still detected in mre11 I158R -6HA at T235 ( Figure 2D, middle panel). These results suggest that mre11 I9R -6HA cannot repair both spontaneous and MMS-induced DSBs due to its defect in MRX assembly. The mre11 I158R -6HA mutant, compared to MRE11-6HA, is partly defective in repairing MMS-induced DSBs.
The results of PFGE ( Figure 2C) are also consistent with those of MMS sensitivity ( Figure 1B) and checkpoint activation ( Figure 2D); the tendency for recovery of MMS-induced replication fork stalls is MRE11-6HA > mre11 I158R -6HA > mre11 I9R -6HA. Due to the presence of forks and bubbles that impede chromosome migration, the incompletely replicated chromosomes did not enter the gel at T55 ( Figure 2C, lanes 8, 14, 17 and 26). Chromosome replication was recovered and almost completed in MRE11-6HA at T235 ( Figure 2C, lane 9), enabling the chromosomes to enter the gel. In contrast, a significant proportion of the chromosomes was never completely replicated in mre11 I9R -6HA and mre11 I158R -6HA at T235 ( Figure 2C, lanes 18 and  27).
Next, we showed that Mre11-6HA coimmunoprecipitated much more V5-tagged CSMs (but not the V5-tagged Smt3 monomers) than Mre11 I9R -6HA or Mre11 I158R -6HA in the absence ( Figure 3A upper panel, lanes 7-9) or presence (Figure 3 upper panel, lanes 10-12) of 0.3% MMS. These results are consistent with those of our two-hybrid assays ( Table 1), indicating that Mre11 and/or the MRX complex preferentially interact with CSMs but not the Smt3 monomer via SIM1 and SIM2.
The immunoblotting results with anti-HA antibodies ( Figure 3 lower and right panel, lanes 7-12) also revealed that Mre11-6HA and Mre11 I158R -6HA, but not Mre11 I9R -6HA, are phosphorylated in response to MMS ( Figure  3 lowest panels, lane 4-6, 10-12). Notably, all these three Mre11-6HA proteins exhibited similar higher mobility patterns in SDS-PAGE ( Figure 3 lowest right panels, lanes 7-12), indicating that neither SIM1 nor SIM2 significantly affects Mre11 SUMOylation (11). From these results, we can infer SUMOylation of Mre11 occurs prior to the assembly of the MRX complex. Consistent with the results of our two-hybrid assays (Table 1), the results here also indicate that Mre11-dependent global SUMOylation is mediated via the Mre11-CSM ensembles rather than the SUMOylated Mre11.
In conclusion, the wild-type vegetative cells can repair both spontaneous and MMS-induced DSBs, initiate DSBinduced global SUMOylation and maintain normal telomere length during vegetative growth. The mre11 I9R mutant fails to assemble the MRX complex, and thus it is defective in all physiological functions of the MRX complex. In contrast, mre11 I158R can repair spontaneous DSBs and maintain normal telomere length, while it is partly defective in repairing MMS-induced DSBs and thus confers higher MMS resistance than mre11 I9R and mre11 . Finally, the mre11 I158R mutant can induce global SUMOylation in response to MMS, but is less efficient than the wild-type cells in promoting SUMOylation of DNA repair proteins.

Mre11 and Mre11 I158R , but not Mre11 I9R , are phosphorylated during meiosis
Notably, before they were transferred to the sporulation medium (i.e. 0-h), all Mre11-6HA proteins in the three strains exhibited the same mobility in an SDS-PAGE gel ( Figure 3A,B, upper panel). Mre11 I9R -6HA exhibited the same mobility throughout sporulation, whereas Mre11-6HA and Mre11 I158R -6HA were post-translationally modified at the 3-h time point and thereafter, migrated more slowly in the SDS-PAGE gel. A dephosphorylation assay was performed as described previously (54) using calf intestinal alkaline phosphatase (CIAP) and 2glycerophosphate (2-GP), a general phosphatase inhibitor. The results reveal that the faster and slower migrating species represent nonphosphorylated and phosphorylated proteins, respectively ( Figure 6D). These results suggest that Mre11 phosphorylation occurs in the context of the MRX complex but is independent of Mre11 SIM2 . Although Mre11 is a SUMOylated protein during vegetative growth ( Figure  3A, lowest right panel), we hardly detected any SUMOylated Mre11-6HA or Mre11 I158R -6HA in meiotic cells by immunoblotting ( Figure 6A,B).

A comparison between mre11 I158R and mre11S
Our results indicate that mre11 I158R allows initiation but not processing and repair of Spo11-induced DSBs similar to sae2 , rad50S and mre11S. The mre11S allele contains two point mutations, P84S and T188I (73). P84 and T188 are evolutionarily conserved. In the S. pombe Mre11-Nbs1 complex crystal structure (58), P93 and S207 (the amino acid equivalents of P84 and T188) reside nearby SIM1 and SIM2, respectively (Supplementary Figure S2). However, neither Mre11 P84S nor Mre11 T188I exhibit apparent defects in two-hybrid interactions with Smt3, Ubc9, Siz2, Xrs2 or Rad50 (Table 1). We found that the S. cerevisiae mre11 P84S -6HA homozygous diploid, like the S. pombe mre11 P93S homozygous diploid (75), displayed no apparent (or modest) defects in sporulation or spore viability (≈89%). In contrast, mre11 T188I -6HA and mre11S-6HA (mre11 P84S, T188I -6HA) are unable to produce any viable spore (Table 2). Therefore, the T188I mutation is the main cause for mre11S (64). As S. cerevisiae T188 and S. pombe S207 are located in a surface loop that contains conserved positively-charged residues critical for DNA binding (Supplementary Figure  S2), the T188I mutation might distort Mre11 DNA binding during processing of meiotic DSBs (58,64,76). Since T188, as for S. pombe S207 (58), is close to SIM2, we speculated that the Mre11 SIM2 -CSM interaction might affect the Mre11 DNA binding during meiosis. Next, we carried out meiotic nuclear spread immunostaining experiments to determine whether Mre11-6HA, Mre11 I9R -6HA and Mre11 I158R -6HA were properly targeted to meiotic chromosomes using the anti-HA antisera ( Figure 7A). It is known that sae2 or rad50S accumulate Mre11 foci, which is not seen in the wild type (27). Our cytology data ( Figure  7B) revealed that the Mre11-6HA foci appeared at the 3.5h sporulation time point in wild-type and sae2 , but only persisted at the 5-h sporulation time point in sae2 . Neither of these two strains examined here formed Mre11 I9R -6HA foci because Mre11 I9R -6HA is unable to assemble the MRX complex. In contrast, Mre11 I158R -6HA foci accumulated in both wild-type and sae2 , indicating that the Mre11 SIM2 -  CSM interaction is dispensable for the Mre11 DNA binding during meiosis. It is of interest to further determine how the Mre11 SIM2 -CSM interaction affects the catalytic function of Mre11 in processing Spo11-induced DSBs.

Mre11 is unlikely to recruit SUMOylated Sae2 for end processing of Spo11-induced DSBs
Sae2 can directly interact with Mre11 (77) and the MRX complex (30) in vitro. There are two putative SUMOylation sites (K97 and K319) in Sae2. The SUMOylation at K97 of Sae2 increases both soluble Sae2 and the MRX function in dsDNA end resection during vegetative growth (21). We postulated that Mre11 SIM2 might recruit SUMOylated Sae2 to reconstitute a functional MRX-Sae2 dsDNA endonuclease (at least) during vegetative growth. However, this hypothesis is not applicable to the repair of Spo11-induced DSBs: (i) Sae2 SUMOylation might not occur in vivo during meiosis because SUMOylated Sae2-3HA was not detected in meiotic cells by immunoblot time course experiments (Supplementary Figure S3). (ii) Both sae2 K97R -3HA (21) and sae2 K97R, K319R -3HA mutants, as compared to wildtype SAE2-3HA diploid cells, underwent normal sporulation and produced (>90%) viable spore ( Table 2). (iii) It has been reported that the phosphorylation of Sae2 is required to initiate resection and to improve the efficiency of resection through cooperation with the MRX complex during meiosis (78,79). Our results also confirm that Sae2-3HA, Sae2 K97R -3HA and Sae2 K97R,K319R -3HA are phosphorylated during meiosis (Supplementary Figure S3).

DISCUSSION
The results in this report indicate that S. cerevisiae Mre11 can non-covalently recruit CSMs (particularly the poly-SUMO chains or their conjugates) to facilitate its assembly and functions during both vegetative growth and meiosis. Mre11 first recruits CSMs via SIM1 to facilitate MRX assembly and/or Mre11 folding, after which, Mre11 recruits CSMs via SIM2 to promote its interaction with the SUMO enzymes (E2/Ubc9 and E3/Siz2). These enzymes then induce global SUMOylation at DSB sites, particularly SUMOylation of DNA repair enzymes. However, the Mre11 SIM2 -CSM interaction does not affect Xrs2dependent Tel1 activation. We also show that the primary defect of the mre11 I158R mutation, as for rad50S, sae2 and mre11S, is to block the resection of the Spo11-induced DSB ends during meiosis. Mre11 SIM1 and Mre11 SIM2 are evolutionarily conserved in S. cerevisiae and S. pombe. It would be interesting to investigate further whether the S. pombe Mre11 SIM1 and Mre11 SIM2 are also important for the assembly of MRN and/or its function in mitosis and meiosis.
Our results support the hypothesis that CSMs can noncovalently prevent premature aggregation by increasing the water solubility of individual protein subunits (8,9) prior to their assembly into a functional MRX complex (10,11,37). Similar to this scenario, SUMOylation of Sae2 increases both soluble Sae2 and the Sae2's function in DNA end resection (21). To coordinate this task in a timely manner, these CSMs or SUMOylation are negatively regulated by deSUMOylation proteases and SUMO-targeted ubiquitin ligases. Consistent with this, mutations in either deSUMOylation protease genes (ulp1 ts , ulp2 ) (16,17) or SUMOtargeted ubiquitin ligase genes (slx5 , slx8 ) (18,19) cause pronounced effects on DSB repair and genomic instability. Accordingly, we suggest that the SIM2-dependent MRX-CSM interaction can provide a regulatory function in regulating MRX function during DSB repair.
A key finding of this study is that SUMOylation of S. cerevisiae Mre11 is unlikely to be required for the formation of the MRX complex and the DSB-induced SUMOylation of DNA repair proteins ( Figure 3A). Mre11-6HA, Mre11 I9R -6HA and Mre11 I158R -6HA all can be SUMOylated during vegetative growth. However, we failed to detect SUMOylated Mre11-6HA by immunoblotting during meiosis ( Figure 6). Our results also indicate that Mre11-6HA and Mre11 I158R -6HA, but not Mre11 I9R -6HA, are phosphorylated and can form MRX complexes during both vegetative growth and meiosis. Because Mre11 I9R -6HA is unable to form an MRX complex, we further inferred that Mre11 SUMOylation and phosphorylation occur before and after Mre11 assembly, respectively. Further investigation will reveal whether phosphorylation of Mre11 is functionally relevant to MRX assembly, DSB-dependent checkpoint activation or global SUMOylation.
SUMOylation of Sae2 increases both soluble Sae2 and the MRX function in DNA end resection during vegetative growth (21). Our results have revealed that SUMOylated Nucleic Acids Research, 2016, Vol. 44, No. 5 2211 Sae2 protein not only was not detected by immunoblotting but also that it is functionally unimportant for meiosis, sporulation and spore viability (Supplementary Figure  S3 and Table 2). Like Mre11, Sae2 is also a phosphorylated protein during meiosis. Phosphorylation of Sae2 Ser-267 by cyclin-dependent kinase 1 (Cdk1) is required to initiate meiotic DSB resection by allowing Spo11 removal from DSB ends (78,79). It is still unclear how phosphorylated Sae2 regulates the Sae2-Mre11 interaction. In S. pombe, Ctp1 is the functional counterpart of Sae2 in S. cerevisiae and CtIP in mammals (80). Sae2, Ctp1 and CtIP share limited homology (approximately 30 amino acids) at their C-terminal RHR motifs (80), which are responsible for dsDNA binding (81). Ctp1 also is referred to as an Nbs1 interacting protein (Nip1) because Nbs1 recruits phosphorylated Ctp1 to DSBs via its binding to the Nbs1 FHA domain of Ctp1 phosphorylated SXT motifs (Ctp1 residues 71-79) (82,83). In contrast, Sae2 interacts with Mre11 but not Xrs2 (77). Furthermore, the SXT motif is not conserved in Sae2. The coupling mechanism between MRX and Sae2 in S. cerevisiae may be different from that of MRX and Ctp1 in S. pombe.
Finally, we previously proposed that the meiosis-specific axial protein Red1 might recruit CSMs to activate Tel1 for Spo11-dependent Hop1 (or Hop1-T318) phosphorylation (11,12). The results in this report indicate that the Red1-CSM complex does not exert this function via an interaction with Mre11 (or the SIM2 in Mre11), because Xrs2dependent Tel1 activation still occurs in the mre11 I158R diploid during meiosis ( Figure 6A). We intend to conduct further studies to decipher the coupling mechanism between Red1 and Tel1.