Rad52 phosphorylation by Ipl1 and Mps1 contributes to Mps1 kinetochore localization and spindle assembly checkpoint regulation

Significance

Rad52 is a well-known factor in homologous recombination. In this study, we discover functions of Rad52 in spindle assembly checkpoint (SAC) regulation and Mps1 localization for chromosome biorientation. Deletion of RAD52 leads to various phenotypes of inaccurate chromosome segregation that are not observed in another homologous recombination-defective rad51Δ mutant. Furthermore, we find that Rad52 is a substrate of mitotic kinases Ipl1/Aurora and Mps1 and that Rad52 phosphorylation by Ipl1 and Mps1 contributes to Mps1 kinetochore localization and SAC regulation. From these findings, we suggest that Rad52 functions as a mediator between Ipl1 and Mps1 in the regulatory pathway of mitosis. Our results provide detailed insights into the molecular basis of chromosome segregation and SAC regulation.

Abstract

Rad52 is well known as a key factor in homologous recombination. Here, we report that Rad52 has functions unrelated to homologous recombination in Saccharomyces cerevisiae; it plays a role in the recruitment of Mps1 to the kinetochores and the maintenance of spindle assembly checkpoint (SAC) activity. Deletion of RAD52 causes various phenotypes related to the dysregulation of chromosome biorientation. Rad52 directly affects efficient operation of the SAC and accurate chromosome segregation. Remarkably, by using an in vitro kinase assay, we found that Rad52 is a substrate of Ipl1/Aurora and Mps1 in yeast and humans. Ipl1-dependent phosphorylation of Rad52 facilitates the kinetochore accumulation of Mps1, and Mps1-dependent phosphorylation of Rad52 is important for the accurate regulation of the SAC under spindle damage conditions. Taken together, our data provide detailed insights into the regulatory mechanism of chromosome biorientation by mitotic kinases.

Chromosome segregation is the most important step during mitosis to maintain genome integrity. Missegregation of chromosomes causes aneuploidy, which induces cell death in microbes and tumors in mammalian cells. Aurora B kinase (Ipl1 in Saccharomyces cerevisiae) and Mps1 are major regulators of chromosome biorientation during mitosis (1–3). Aurora B kinase localizes between sister kinetochores (4) and phosphorylates target proteins at the kinetochores in a distance-dependent manner to monitor chromosome biorientation (5, 6). Mps1 is known as an activator of the spindle assembly checkpoint (SAC) under conditions of spindle damage, but its function in regulating unperturbed mitosis has recently been revealed (2, 3). To function properly, Mps1 should be localized to the kinetochores by Aurora B kinase activity (7–9). However, the precise mechanism for the recruitment of Mps1 to the kinetochores is not yet known.

The mitotic checkpoint is a cell cycle checkpoint that delays mitosis to allow for accurate chromosome segregation and cell division. In the budding yeast S. cerevisiae, the mitotic checkpoint is composed of two pathways (10). First, the Mad1-dependent pathway, known as the SAC, is activated by Mps1 and suppresses the APC/C complex, which is an E3 ubiquitin ligase for the degradation of Pds1 (securin homolog in S. cerevisiae) and Clb2 (cyclin B in S. cerevisiae) (11, 12). Mps1-dependent phosphorylation of Spc105 (Knl1 homolog in S. cerevisiae) accumulates the components of the SAC such as Mad1, Mad2, and Bub1 to the kinetochores (13, 14). Subsequently, Mad2 forms the mitotic checkpoint complex (MCC) with Cdc20 (15), and Cdc20 in MCC is degraded in a Mad2- and APC/C-dependent manner to activate the SAC (16). Second, the Bub2-dependent pathway, known as the spindle position checkpoint (SPOC), suppresses the release of Cdc14 to inhibit mitotic exit (10, 17, 18). Cdc14 is regulated by the Cdc14 early anaphase release (FEAR) network and the mitotic exit network (MEN) (19). When the spindle is arranged incorrectly, Bub2/Bfa1 suppresses Ras-like GTPase Tem1, which is a positive regulator of MEN to prevent anaphase promotion (20).

In this study, we reveal functions of Rad52 in cell growth regulation. Although the roles of Rad52 in homologous recombination and DNA damage repair have been known for a while, its other functions have not yet been discovered. Here we find that Rad52 is a substrate of Ipl1/Aurora and Mps1. In addition, Rad52 regulates Mps1 recruitment to the kinetochores and plays an important role in the proper operation of the SAC. In total, our results suggest that Rad52 is a regulator of precise chromosome segregation in mitosis.

Results

Deletion of RAD52 Leads to Defects in Accurate Chromosome Segregation.

To observe the morphology of chromosome segregation during vegetative growth, we used GFP-tagged Ndc80 as a marker for total kinetochores. All of the kinetochores in wild-type cells accumulated at both spindle poles (Fig. 1A, Left). Interestingly, in rad52Δ cells, we observed kinetochore declustering during segregation, which are the hallmark of incorrect microtubule attachment and improper chromosomes segregation (21, 22). The ratio of cells with kinetochore declustering was significantly increased in rad52Δ cells (Fig. 1A, Right). Deletion of RAD51, which is another key factor for homologous recombination, did not induce kinetochore declustering. Because rad52Δ cells exhibited increased kinetochore declustering, we speculated that Rad52 influences the accurate regulation of chromosome segregation.

Fig. 1.

rad52Δ cells show inaccurate chromosome segregation. (A) Analysis of kinetochore morphology during vegetative growth. Ndc80-GFP and Spc29-RFP were used as markers for the kinetochores and spindle poles, respectively. Fluorescence intensity was measured along yellow lines on the images (Left). White arrows on the image and black arrows on the relative fluorescence intensity graph indicate kinetochore declustering that did not accumulate on the spindle poles properly. (Scale bars, 4 μm.) The percentage of cells exhibiting different kinetochore morphology is shown on the Right. P values were determined by Student’s t test (**P < 0.01; ns, not significant). (B) Measurement of chromosome inheritance fidelity during vegetative growth by pRS415 vector loss assay. (C) Measurement of chromosome segregation fidelity during mitosis. The indicated strains synchronized by α-factor treatment for 3 h were released to fresh YPD media and samples were taken every 15 min from 60 min to 105 min after release from α-factor. (D) Colocalization of Rad52 with Ipl1. GFP-tagged Ipl1 and RFP-tagged Rad52 were endogenously expressed from their native promoters. After cells in log phase were incubated with galactose for 2 h, 15 μg/mL nocodazole and 0.003% MMS were treated for 2 h. (Scale bars, 2 μm.) The percentage of cells exhibiting colocalization of Rad52 with Ipl1 under the indicated conditions is shown on the Right. P values were determined by Student’s t test (**P < 0.01; ns, not significant). (E) Confirmation of Rad52 localization to the kinetochores by ChIP assay. Rad52-HA ChIP assays were performed for CEN3 and noncentromeric DNA (nonCEN). Rad52-HA column of CEN3 was used as a standard to calculate relative fold enrichment of each column. P values were determined by the one-sample t test (**P < 0.01). All data in this figure represent the mean ± SD of triplicate experiments.

Yeast minichromosomes with yeast centromere sequences are transmitted with high fidelity by the chromosome segregation machinery, and biorientation of minichromosomes is impaired by the inactivation of the regulatory system for chromosome inheritance (23, 24). We measured chromosome inheritance fidelity during vegetative growth by using the pRS415 vector, which has the yeast CEN6 sequence. As shown in Fig. 1B, rad52Δ cells lost pRS415 much faster than wild-type and rad51Δ cells, suggesting that the regulatory system for chromosome inheritance is not efficiently controlled in rad52Δ cells. This result is consistent with a previous study that reports high frequency of mitotic chromosome loss in rad52Δ cells (25).

To find direct evidence of chromosome missegregation, we checked the fidelity of chromosome segregation during mitosis by using a CEN5-GFP strain that contains CEN5-(tetO2)₁₁₂ and tetR-GFP (26). In addition, we measured the amount of Pds1 after release from α-factor arrest to precisely monitor anaphase onset, which is triggered at ∼60 min after release from α-factor arrest (SI Appendix, Fig. S1A). Most of wild-type and rad51Δ cells properly segregated sister chromatids with each of spindle poles that separately moved to the mother and daughter cell (Fig. 1C). In rad52Δ cells, however, the ratio of cells containing missegregated CEN5-GFP was remarkably increased at 75 min, which is during anaphase, suggesting that deletion of RAD52 causes misregulation of chromosome segregation. Interestingly, we observed that Rad52 colocalizes with Ipl1, which is the major regulatory kinase for the regulation of chromosome segregation, before the onset of anaphase (Fig. 1D). To synchronize the cell phase before anaphase onset, pds1-mdb was expressed under the control of a GAL promoter. pds1-mdb is an APC^CDC20-dependent degradation-defective Pds1 mutant with amino acid substitution in the destruction box; therefore, the expression of pds1-mdb induces metaphase arrest and suppresses entry into anaphase (11, 27). Approximately 14% of cells exhibited colocalization of Ipl1 and Rad52 under normal conditions, and the colocalization ratio was increased to 28% by treatment of microtubule-depolymerizing drug nocodazole (Fig. 1D). This increase in colocalization of Ipl1 and Rad52 was not detected under methylmethane sulfonate (MMS) treatment, even though the Rad52 foci were observed more under MMS-treated conditions than normal conditions. This result suggests that the disruption of microtubule–kinetochore attachment stimulates Rad52 accumulation at the kinetochores. The localization of Rad52 to the kinetochores was also confirmed by chromatin immunoprecipitation (ChIP) assay. Rad52-HA ChIP followed by quantitative real-time PCR (qPCR) revealed that Rad52 bound to the centromere locus (Fig. 1E). Taken together, we hypothesized that Rad52 is closely involved in the accurate regulation of chromosome segregation.

Inaccurate Chromosome Segregation in rad52Δ Cells Is Not Related to Loss of Homologous Recombination Activity.

In addition to experiments using rad51Δ cells, we further examined the possibility that loss of homologous recombination activity may cause inaccurate regulation of chromosome segregation using the homologous recombination-defective mutants of Rad52. rad52[F316A,Y376A] and rad52[L56F] are defective mutants of Rad51-dependent and Rad51-independent homologous recombination, respectively (28, 29). As previously reported, these mutants failed to recover DNA damage caused by MMS, zeocin, and phleomycin (SI Appendix, Fig. S2A). Contrary to rad52Δ cells, however, rad52[F316A,Y376A] and rad52[L56F] cells did not exhibit a significant increase in the ratio of cells with kinetochore declustering (SI Appendix, Fig. S2B). We also examined the precise chromosome missegregation ratio using CEN5-GFP strains. After anaphase onset, the ratios of rad52[F316A,Y376A] and rad52[L56F] cells with missegregated CEN5-GFP were not significantly different from that of wild-type cells (SI Appendix, Figs. S1B and S2C). These results suggest that the homologous recombination activity of Rad52 is not required for the accurate regulation of chromosome segregation.

Next, we checked the possibility that loss of Rad52 may increase spontaneous occurrence of DNA damage, which unexpectedly leads to chromosome missegregation. To measure the occurrence of intracellular DNA damage in wild-type and rad52Δ cells, we used Rad53 phosphorylation in cells as a marker for endogenous DNA damage (30). Rad53 is a regulatory kinase for the DNA double-strand break repair pathway. When DNA damage occurs, the DNA damage checkpoint kinase Mec1 phosphorylates Rad53 to activate the DNA damage checkpoint and repair pathway (31). rad52Δ cells exhibited a similar Rad53 phosphorylation pattern to that of wild-type cells both in the presence and absence of MMS (SI Appendix, Fig. S3A), indicating that there is no difference in intracellular DNA damage occurrence between wild-type and rad52Δ cells. To exclude the possibility that mild DNA damage might not be detected by this method, we examined the sensitivity of Rad53 phosphorylation using HO endonuclease, a sequence-specific double-strand nuclease of yeast used in mating type switch. Unlike MMS, the expression of HO endonuclease results in a single DNA double-strand break, which is repaired by the Rad53-mediated DNA repair pathway (32). While Rad53 phosphorylation was clearly induced by HO expression in both wild-type and rad52Δ cells, Rad53 phosphorylation was not detected in rad52Δ cells without HO expression as in wild-type cells (SI Appendix, Fig. S3B). We also compared the occurrence of spontaneous DNA damage in wild-type and rad52Δ cells by checking the foci formation of Rfa1 and the protein amount of Sml1, which are used as the indicators of intracellular DNA damage occurrence (33, 34). Consistent with Rad53 data, rad52Δ cells did not show any significant difference in the foci formation of Rfa1 and the protein amount of Sml1 compared with wild-type cells (SI Appendix, Fig. S3 C and D). These results suggest that spontaneous DNA damage occurrence and accumulation are negligible, if any, in rad52Δ cells during vegetative growth and are not the cause of the improper chromosome segregation in rad52Δ cells.

According to the findings by Mitra et al. (35), loss of Rad52 reduces the level of the kinetochore protein CENP-A^CaCse4, resulting in a disruption of the kinetochore structure in Candida albicans. To check whether the improper chromosome segregation in rad52Δ cells is caused by a disruption of the kinetochore structure, we performed experiments with Cse4, a S. cerevisiae homolog of CENP-A^CaCse4. Unlike CENP-A^CaCse4 in RAD52-deleted C. albicans, the fluorescence intensity of centromere-localized Cse4 was not different between wild-type and rad52Δ cells (SI Appendix, Fig. S4A), and the protein level of Cse4 in rad52Δ cells was also similar to that of wild-type cells (SI Appendix, Fig. S4B). Additionally, in contrast to CENP-A^CaCse4, the centromere-binding affinity of Cse4 was not altered by deletion of RAD52 in S. cerevisiae (SI Appendix, Fig. S4C). These observations suggest that, unlike C. albicans, the improper chromosome segregation in rad52Δ cells is not caused by a disruption of the kinetochore structure in S. cerevisiae. Taken together, these results support our hypothesis that, apart from the previously reported functions, Rad52 is closely related to the accurate regulation of chromosome segregation.

SAC Does Not Efficiently Suppress Anaphase Promotion Under Absence of Rad52.

To confirm our hypothesis, we examined cell viability under nocodazole treatment. Mitotic checkpoint-defective mutants such as mad1Δ and bub2Δ cells showed high sensitivity to nocodazole (Fig. 2A). Interestingly, rad52Δ cells also showed high sensitivity to nocodazole similar to that of mad1Δ and bub2Δ cells. We also checked whether rad52Δ cells show the rereplication of chromosomes under nocodazole treatment. Consistent with a previous report (10), mad1Δ and bub2Δ cells accumulated DNA contents higher than 2C under nocodazole treatment while wild-type cells stably stayed in G2/M phase under the same conditions (Fig. 2B). Although the timing of rereplication of chromosomes was delayed for about 60 min compared with mad1Δ and bub2Δ cells, rad52Δ cells could not stay in G2/M phase and their DNA contents were sequentially increased over 2C. This observation suggests that rad52Δ cells also show the rereplication of chromosomes under prolonged nocodazole treatment. Taken together with the above results, it seems obvious that rad52Δ cells show the phonotypes resulting from improper regulation of the SAC.

Fig. 2.

Rad52 is required for proper chromosome segregation. (A) Cell viability assay under nocodazole treatment. Cell viability was calculated by normalizing the colony numbers of nocodazole-treated cells to those of untreated cells. P values were determined by Student’s t test (**P < 0.01). (B) Measurement of DNA content by flow cytometry. The indicated strains were synchronized by 150 μM α-factor treatment for 3 h, and samples were taken every 30 min after release to YPD containing 15 μg/mL nocodazole. The 1C and 2C indicate single and double DNA haploid content, respectively. (C) Measurement of chromosome segregation fidelity after release from nocodazole arrest. The indicated strains with CEN5-GFP and Spc29-RFP signals were synchronized by 150 μM α-factor treatment for 3 h and released to YPD containing 15 μg/mL nocodazole for 1 h. After nocodazole washout, samples were taken every 20 min. (Scale bars, 2 μm.) The percentage of cells exhibiting normal and abnormal CEN5 alignment in the indicated strains are shown on the Right.

To more precisely test the regulation of chromosome segregation under absence of Rad52, we treated cells with nocodazole to depolymerize microtubules and chased the recovery of chromosome alignment during the further incubation after nocodazole washout. Because nocodazole treatment efficiently causes the destruction of mitotic spindles, CEN5-GFP signal was shown as one dot, even though spindle poles were duplicated and separated (SI Appendix, Fig. S5). In wild-type cells, during the further incubation after nocodazole washout, CEN5-GFP signal was efficiently captured to spindle poles and separated to two dots (SI Appendix, Fig. S5, Upper). Subsequently, CEN5-GFP signals were normally segregated to each end of cells. However, it was frequently observed that CEN5-GFP signal in rad52Δ cells was not separated to two dots close to spindle poles after nocodazole washout (SI Appendix, Fig. S5, Lower). Even more remarkably, although proper alignment of CEN5 was not established, spindle poles were moved to the ends of cells without any significant delay.

To further confirm this phenomenon, we examined the ratio of cells with different CEN5-GFP signal patterns, according to the time after release from nocodazole treatment. In all tested strains, more than 70% of cells showed improper alignment of CEN5-GFP at 0 min after nocodazole washout (Fig. 2C). Eventually, wild-type cells recovered proper CEN5-GFP alignment and a large portion of cells with proper CEN5-GFP alignment sequentially exhibited chromosome segregation. In contrast, improper CEN5-GFP alignment in rad52Δ cells was not efficiently recovered even at 60 min after nocodazole washout. Nevertheless, consistent with time-lapse data of CEN5-GFP (SI Appendix, Fig. S5, Lower), rad52Δ cells with improper CEN5-GFP alignment exhibited movement of spindle poles to the ends of cells with similar kinetics to those of wild-type cells with proper CEN5-GFP alignment (Fig. 2C). If the SAC is functioning properly, cells showing improper CEN5-GFP alignment will be arrested before the progression of sister-chromatids separation. Thus, this observation suggests that the SAC is not functioning properly without Rad52. Notably, loss of homologous recombination activity by deletion of RAD51 did not affect proper recovery of CEN5-GFP alignment after nocodazole washout. As expected, the defect in the SAC by deletion of MAD1 clearly caused monotelic separation of CEN5-GFP during the additional incubation without nocodazole. These results suggest that, similarly to Mad1, Rad52 contributes to the proper operation of the SAC.

To reinforce our results, we examined the degradation of Cdc20 under spindle damage conditions. As previously described (16), Cdc20 was rapidly degraded in wild-type and rad51Δ cells after release from α-factor arrest to fresh medium containing nocodazole (Fig. 3A). However, the protein level of Cdc20 was considerably maintained in rad52Δ cells until 240 min. Next, we analyzed the degradation kinetics of Pds1 under nocodazole treatment. While wild-type and rad51Δ cells maintained a high level of Pds1 until 240 min, the Pds1 level in rad52Δ cells decreased remarkably after 150 min (Fig. 3B). Interestingly, a SAC-defective mutant, mad1Δ, showed more rapid degradation of Pds1 than rad52Δ cells, suggesting that Rad52 affects the maintenance of sufficient SAC activity rather than initial SAC activation. Taken together, we conclude that Rad52 has an undefined role in the regulation of chromosome segregation and the SAC.

Fig. 3.

Rad52 is necessary to maintain SAC activity. (A) Degradation kinetics of Cdc20 under nocodazole treatment. Samples were taken at the indicated time intervals. Hexokinase was used as a loading control. (B) Degradation kinetics of Pds1 under nocodazole treatment. An immunoblot assay was performed using an anti-myc antibody (Left). The highest value in each trial was used as the 100% standard to normalize the relative amounts of Pds1 (Right). All data in this figure represent the mean ± SD of triplicate experiments.

Rad52 Is a Substrate of Ipl1 and Mps1.

The SAC-related proteins and regulatory proteins of chromosome segregation are controlled by mitotic kinases such as Ipl1 and Mps1 (1–3). Thus, we tested whether Rad52 is regulated by these mitotic kinases. Rad52 was clearly separated to a slow- and a fast-migrating band on SDS/PAGE (Fig. 4A). By using the Phos-tag assay and the λ-phosphatase treatment assay, we confirmed that the slow-migrating band is a phosphorylated form of Rad52. Rad52 has five Ipl1 consensus residues, which are serine or threonine in (R/K)X(S/T)(I/L/V) (36) (SI Appendix, Fig. S6A). A non–Ipl1-phosphorylatable mutant of Rad52 {Rad52-5A; Rad52[S86A,T96A,S136A,T349A,S374A]} exhibited a significant decrease in phosphorylation (Fig. 4A). Rad52-5A could repair DNA damage generated by DNA damage agents (SI Appendix, Fig. S6B), suggesting that alanine substitution of Ipl1 consensus sequences on Rad52 does not cause severe structural alterations leading to an enzymatic dysfunction.

Fig. 4.

Rad52 is phosphorylated by Ipl1 and Mps1. (A) Analysis of Rad52 phosphorylation by SDS/PAGE. The phosphorylation state of wild-type Rad52 and Rad52[S86A,T96A,S136A,T349A,S374A] (Rad52-5A) was examined by SDS/PAGE in the presence (Upper) or absence (Lower) of 20 μM Phos-tag. The relative ratio of phosphorylated (P-Rad52) to dephosphorylated forms of Rad52 on the 20 μM Phos-tag SDS/PAGE is shown Below each lane. (B) Analysis of cell cycle-dependent phosphorylation of Rad52. rad52Δ cells expressing Rad52 or Rad52-5A were synchronized using 0.2 M hydroxyurea (HU) treatment for 3 h, and samples were taken every 30 min after release to fresh SC −Ura medium. The relative ratio of phosphorylated-to-dephosphorylated forms of Rad52 is shown Below each lane. (C) In vitro kinase assay using Ipl1. An in vitro kinase assay was performed with purified proteins from E. coli. An immunoblot assay was conducted with an anti-GST antibody. (D) In vitro kinase assay using four revertants of Rad52. Rad52[S136A,T349A,S374A] (S86A,T96A revertant), Rad52[S86A,T96A,T349A,S374A] (S136A revertant), Rad52[S86A,T96A,S136A,S374A] (T349A revertant), and Rad52[S86A,T96A,S136A,T349A] (S374A revertant) were made by reverting mutated residues from Rad52-5A. (E) In vitro kinase assay using Mps1. An in vitro kinase assay was performed with Rad52 purified from E. coli and Mps1 immunoprecipitated from S. cerevisiae. (F) In vitro kinase assay using four revertants of Rad52. An immunoblot assay was performed using an anti-GST antibody for Rad52 and a rabbit anti-mouse IgG antibody for Mps1. (G) Analysis of Rad52 phosphorylation in ipl1-321 cells. The indicated strains were grown in 2% raffinose-containing YP media and synchronized by α-factor treatment for 3 h at 25 °C. A total of 2% galactose was added to the cell cultures 30 min before release from α-factor arrest. Synchronized cells were released to 2% galactose-containing YP media with nocodazole for 2 h at 25 °C or 37 °C. The DNA content data were analyzed by flow cytometry (Upper). The 1C and 2C indicate single and double DNA haploid content, respectively. The phosphorylation state of Rad52 in the indicated strains was examined by SDS/PAGE and an anti-HA immunoblot assay (Lower). The relative ratio of phosphorylated-to-dephosphorylated forms of Rad52 is shown Below each lane. pds1-mdb tagged with RFP was expressed under the control of a GAL promoter and blotted with an anti-RFP antibody. (H) Analysis of Rad52 phosphorylation in mps1-as1 cells. Cell synchronization was performed as described in G. Synchronized cells were released to 2% galactose-containing YP media with nocodazole for 2 h with or without 5 μM 1NM-PP1. The DNA content data and the phosphorylation state of Rad52 were analyzed as described in G and are shown Upper and Lower, respectively.

Notably, phosphorylation of Rad52 was highly increased during metaphase and decreased at 90 min (Fig. 4B), which is considered the cell phase after the onset of anaphase. However, phosphorylation of Rad52-5A was not changed during cell cycle progression, suggesting that mitotic kinases phosphorylate Rad52 on five Ipl1 consensus residues. To check whether Ipl1 is a kinase for Rad52, we examined in vitro phosphorylation of Rad52 by Ipl1. Ipl1 phosphorylated Rad52 on the Ipl1 consensus residues, whereas the kinase-dead Ipl1 with K133R (37) could not (Fig. 4C). To determine the exact phosphorylation site, each mutated residue of Rad52-5A was reverted to the original residue. Among the revertants, only Rad52[S86A,T96A,S136A,T349A] was phosphorylated by Ipl1 (Fig. 4D), suggesting that Ipl1 is a kinase for Rad52 and the target residue is S374.

Because rad52Δ cells failed to maintain sufficient activity of the SAC under spindle damage conditions (Fig. 3), we examined whether Rad52 is also a substrate of Mps1 using an in vitro kinase assay. As shown in Fig. 4E, Mps1 phosphorylated Rad52, but the kinase-dead Mps1 with D580A (38) could not. Interestingly, Rad52-5A was not phosphorylated by Mps1, suggesting that the target residue of Mps1 is within the five Ipl1 consensus residues. By using four revertants of Rad52-5A, we observed that S86 or T96 was strongly phosphorylated and S136 was weakly phosphorylated by Mps1 (Fig. 4F). To confirm that Rad52 is a target of Ipl1 and Mps1 in vivo, we tested the phosphorylation of Rad52 in a temperature-sensitive ipl1-321 mutant (39) and an ATP analog-sensitive mps1-as1 mutant (40). Additionally, to precisely arrest the cell cycle in metaphase, in which Rad52 is highly phosphorylated, pds1-mdb expression was combined with nocodazole treatment. Rad52 was highly phosphorylated in metaphase, but when the kinase activities of Ipl1 and Mps1 were suppressed, the phosphorylation ratio of Rad52 was significantly decreased (Fig. 4 G and H). Taken together, these results suggest that Rad52 is a substrate of Ipl1 and Mps1.

Human Rad52 has a similar amino acid sequence and protein structure to yeast Rad52 (41). Thus, we checked whether human Rad52 is a substrate of human Aurora B kinase and human Mps1. Human Rad52 was efficiently phosphorylated by Aurora B kinase (SI Appendix, Fig. S6C). Interestingly, yeast Rad52 was also phosphorylated by Aurora B kinase, and Ipl1 could phosphorylate human Rad52. In addition, human Mps1 phosphorylated human Rad52 and yeast Rad52, similarly to Aurora B kinase (SI Appendix, Fig. S6D). These data suggest that Rad52 phosphorylation by Ipl1 and Mps1 is also conserved in human cells. Therefore, it is possible that the functions of Rad52, which are regulated by Ipl1- and Mps1-dependent phosphorylation, are conserved from yeast to humans.

Mps1-Dependent Phosphorylation of Rad52 Is Required to Maintain Sufficient Activity of the SAC.

Next, we tested whether the SAC is regulated by Mps1-dependent phosphorylation of Rad52. RAD52 cells with wild-type Rad52 maintained high levels of Pds1 in nocodazole-treated media (Fig. 5A). However, rad52-3A cells with Rad52[S86A,T96A,S136A], which is not phosphorylated by Mps1, and rad52-5A cells with Rad52[S86A,T96A,S136A,T349A,S374A], which is not phosphorylated by either Mps1 or Ipl1, showed a rapid decrease in Pds1 levels after 150 min of nocodazole treatment. This observation suggests that Mps1-dependent phosphorylation of Rad52 is important to maintain sufficient activity of the SAC under spindle damage conditions.

Fig. 5.

Mps1-dependent phosphorylation of Rad52 is required to maintain sufficient SAC activity during prolonged nocodazole treatment. (A) Degradation kinetics of Pds1 under nocodazole treatment. RAD52, rad52-3A, and rad52-5A indicate rad52Δ cells with integrated DNA sequences encoding each Rad52 variant. An immunoblot assay was performed using an anti-myc antibody (Upper). The highest value in each trial was used as the 100% standard to normalize the relative amount of Pds1 (Lower). (B) Recovery fidelity of kinetochore–spindle connections after release from nocodazole arrest. Arrows on the images indicate scattered kinetochores (Upper). (Scale bars, 2 μm.) Cells were sampled every 20 min after nocodazole washout. The percentage of cells exhibiting kinetochore declustering are shown (Lower). (C) Cell viability assay under nocodazole treatment. RAD52, rad52-3A, and rad52-5A cells were incubated in 15 μg/mL of nocodazole-treated media for the indicated times. Nocodazole-treated cells were aligned on a YPD plate using a tetrad dissection microscope. Viability was calculated by normalizing the number of colonies to the total number of aligned cells. P values were determined by Student’s t test (*P < 0.05; ***P < 0.005; ns, not significant). All data in this figure represent the mean ± SD of triplicate experiments.

Because nocodazole depolymerizes microtubules, the chromosomes in nocodazole-treated cells cannot maintain their proper localization and are scattered in the nucleoplasm. After removing nocodazole, the microtubules are regenerated and connected to the adjacent kinetochores. During the restoration of the connection between the microtubule and the kinetochore, incorrect connections are formed. For proper sister-chromosome separation, incorrect attachments must be repaired by Ipl1 and the SAC pathway (42). To check the ability of cells with Rad52 mutants to repair incorrect attachments, we examined the kinetochore morphology and chromosome separation after depolymerizing the microtubules with nocodazole treatment. Approximately 60% of the nocodazole-treated cells showed scattered chromosomes in the nucleoplasm (Fig. 5B). As expected, the chromosomes in cells with wild-type Rad52 segregated to the correct spindle poles. In contrast, scattered chromosomes in rad52-3A and rad52-5A cells predominantly led to kinetochore declustering, and the ratio of rad52-3A and rad52-5A cells with kinetochore declustering was not decreased until 80 min after the removal of nocodazole, suggesting that the spindle–kinetochore interaction is not efficiently recovered in rad52-3A and rad52-5A cells. Furthermore, when incubated with nocodazole, the viability of the rad52-3A and rad52-5A cells was significantly decreased compared with cells with wild-type Rad52 (Fig. 5C). These data indicate that Mps1-dependent phosphorylation of Rad52 regulates the recovery of accurate spindle–kinetochore interactions when spindle damage occurs.

Ipl1-Dependent Phosphorylation of Rad52 Is Required for Mps1 Recruitment to the Kinetochores.

Although rad52-3A and rad52-5A cells showed a similar defect in the SAC regulation (Fig. 5), we observed some different phenotypes between rad52-3A and rad52-5A cells. Interestingly, chromosome segregation was properly regulated in rad52-3A cells, whereas the ratio of chromosome missegregation was significantly increased in rad52-5A cells at 75 min after release from α-factor arrest (Fig. 6A and SI Appendix, Fig. S1B). This observation suggests that Mps1-dependent phosphorylation is important for the maintenance of the SAC activity under spindle damage conditions but not for the regulation of accurate chromosome segregation. To examine whether the defect in Ipl1-dependent phosphorylation of Rad52 leads to improper regulation of chromosome segregation, we checked the fidelity of chromosome segregation in cells expressing Rad5[S374A], which is a non–Ipl1-phosphorylatable Rad52 mutant. As shown in Fig. 6A, the ratio of chromosome missegregation was remarkably increased in rad52[S374A] cells compared with RAD52 and rad52-3A cells. Taken together with the result that Rad52 is a substrate of Ipl1 (Fig. 4D), these data support an idea that the Ipl1-dependent function of Rad52 is an upstream process of the Mps1-dependent function for the accurate regulation of mitosis, which includes proper chromosome segregation during unperturbed cell cycle and proper operation of the SAC under spindle damage conditions. Consistent with this idea, rad52[S374A] cells also could not maintain sufficient SAC activity similar to rad52-3A and rad52-5A cells (SI Appendix, Fig. S7A).

Fig. 6.

Ipl1-dependent phosphorylation of Rad52 facilitates the accumulation of Mps1 at the kinetochores. (A) Measurement of chromosome segregation fidelity during mitosis. The indicated strains synchronized by α-factor treatment for 3 h were released to fresh YPD media and samples were taken every 15 min from 60 min to 105 min after release from α-factor. (B) Analysis of Mps1 accumulation at the kinetochores under nocodazole treatment. Cells in log phase were incubated in galactose-containing YP media for 2 h, followed by 15 μg/mL nocodazole treatment for 2 h. (Scale bars, 4 μm.) P values were determined by Student’s t test (Upper Right). The fluorescence intensity was calculated by subtracting background intensity from Mps1-GFP signal intensity (Lower Right). A box plot was represented with whiskers from the 5th to the 95th percentile, and the data were normalized to the median of RAD52 cells (n > 200). P values were determined by Mann–Whitney u test (***P < 0.005; ns, not significant). (C) Confirmation of Mps1 accumulation at the kinetochores by ChIP assay. Mps1-TAP ChIP assays were performed in the indicated strains for CEN3 and noncentromeric DNA (nonCEN). Wild-type RAD52 column of CEN3 was used as a standard to calculate relative fold enrichment of each column. P values were determined by one-sample t test (*P < 0.05; ***P < 0.005; ns, not significant). (D) Degradation kinetics of Pds1 under expression of Ndc80-Mps1. The indicated strains were grown in 2% raffinose-containing YP media and synchronized by α-factor treatment for 3 h. A total of 2% galactose was added to the cell cultures 30 min before release from α-factor arrest. Synchronized cells were released to 2% galactose-containing YP media (Left). The highest value in each trial was used as the 100% standard to normalize the relative amount of Pds1 (Right). All data in this figure represent the mean ± SD of triplicate experiments.

Given that Rad52 is a substrate shared by Ipl1 and Mps1 (Fig. 4) and the Ipl1-dependent function of Rad52 is an upstream process of the Mps1-dependent function for the accurate regulation of mitosis (Fig. 5 and SI Appendix, Fig. S7A), we hypothesized that Rad52 may act as an Ipl1-dependent Mps1 regulator. To examine whether Rad52 itself has a physical interaction with Mps1 as a direct regulator of Mps1, we performed a GST pull-down assay. Notably, Mps1 was efficiently coprecipitated with purified Rad52 (SI Appendix, Fig. S7B), suggesting that Rad52 is an Mps1-binding protein. Furthermore, the kinetochore localization of Rad52 was also affected by the Ipl1-dependent phosphorylation. Rad52[S374A] was not efficiently accumulated at the kinetochores under spindle damage conditions (SI Appendix, Fig. S7C). This result was also confirmed in a temperature-sensitive ipl1-321 mutant. When the kinase activity of Ipl1 was suppressed, Rad52 accumulation at the kinetochores was significantly decreased under spindle damage conditions (SI Appendix, Fig. S7D). These data raise the possibility that Ipl1-dependent phosphorylation of Rad52 affects the kinetochore accumulation of Mps1. To check this possibility, we investigated whether the nonphosphorylatable mutants of Rad52 affect the accumulation of Mps1 at the kinetochores. Because Mps1-GFP signals could not be detected in our system under normal conditions (SI Appendix, Fig. S8A), we treated cells with nocodazole to induce Mps1 accumulation at the kinetochores during the suppression of anaphase onset by pds1-mdb expression. Interestingly, although rad52-3A cells could not sufficiently maintain the SAC activity (Fig. 5A), Mps1 efficiently accumulated at the kinetochores in rad52-3A cells as well as in RAD52 cells (Fig. 6B, Left). In contrast, Mps1 accumulation at the kinetochores in rad52-5A and rad52[S374A] cells was decreased to ∼50% of that in RAD52 cells (Fig. 6B, Upper Right). Moreover, the fluorescence intensity of Mps1-GFP spots at the kinetochores was also significantly decreased in rad52-5A and rad52[S374A] cells compared with RAD52 and rad52-3A cells (Fig. 6B, Lower Right). Because the cellular amount of Mps1 protein was not different among RAD52 mutants (SI Appendix, Fig. S8B), the decrease in Mps1-GFP signal in rad52-5A and rad52[S374A] cells is assumed to be caused by inefficient accumulation of Mps1 at the kinetochores. To precisely measure the kinetochore accumulation of Mps1, we performed the ChIP assay under nocodazole-treated conditions. Mps1-TAP ChIP followed by qPCR in the nonphosphorylatable Rad52 mutants revealed that Mps1 was significantly less accumulated at the kinetochores in rad52-5A and rad52[S374A] cells compared with RAD52 and rad52-3A cells (Fig. 6C). Taken together, our results suggest that Ipl1-dependent phosphorylation of Rad52 is required to regulate Mps1 recruitment to the kinetochores.

Because the defect in Ipl1-dependent phosphorylation of Rad52 causes insufficient accumulation of Mps1 at the kinetochores under the SAC-activated conditions, we asked whether the forced accumulation of Mps1 at the kinetochores compensates for the effect of non–Ipl1-phosphorylatable Rad52. To force the localization of Mps1 to the kinetochores, Mps1 was tethered to a kinetochore protein Ndc80 and this fusion protein (Ndc80–Mps1) was conditionally expressed under the control of a GAL promoter. Ndc80–Mps1 fusion protein was well expressed in galactose-containing media and properly localized to the kinetochores (SI Appendix, Fig. S8C). Consistent with previous reports (8, 43), the expression of kinetochore-tethered Mps1 stimulated SAC activation (Fig. 6D). In contrast, the expression of kinetochore-tethered kinase-dead Mps1 {Mps1[D580A]} did not stimulate the SAC, suggesting that SAC activation is mediated by the kinase activity of Mps1 but not caused by the unexpected side effects of the Ndc80–Mps1 fusion protein. Notably, the expression of kinetochore-tethered Mps1 in rad52[S374A] cells effectively suppressed the degradation of Pds1, as in wild-type cells. This result demonstrates that artificial recovery of Mps1 localization compensates for insufficient maintenance of SAC activity caused by non–Ipl1-phosphorylatable mutation of Rad52.

Discussion

In this study, we show that Ipl1 regulates Rad52 to facilitate Mps1 localization to the kinetochores and the kinetochore-localized Mps1 phosphorylates Rad52 to sufficiently maintain SAC activity under spindle damage conditions. Given that Mps1 promotes chromosome biorientation in a SAC-independent manner (2), appropriate localization of Mps1 by the regulatory proteins including Rad52 is important for accurate regulation of mitosis even under absence of spindle stresses. Thus, according to our findings, Rad52 appears to regulate mitosis by the following steps: (i) During mitosis, Rad52 is phosphorylated and is accumulated at the kinetochores in a subset of cells to properly orchestrate an undefined step in chromosome segregation, which may or may not be connected to Mps1. (ii) If the spindle–kinetochore attachment is not properly established, Ipl1 highly phosphorylates Rad52 to increase Rad52 accumulation at the kinetochores. (iii) The kinetochore-accumulated Rad52 stimulates Mps1 recruitment to the kinetochores. (iv) Highly accumulated Mps1 phosphorylates Rad52 to promote robust signaling through the SAC. In addition, we also provide evidence that human Rad52 is phosphorylated by Aurora B kinase and human Mps1 (SI Appendix, Fig. S6 C and D). Because the kinetochore structure and the SAC pathway are well conserved from yeast to humans, it is possible that human Rad52 may act as a mitosis regulator in human cells.

Interestingly, the Mps1 target residues on Rad52 are within the Ipl1 consensus residues (Fig. 4F). Overlapping target residues of Ipl1 and Mps1 have already been described in Ndc80 (44, 45). It is likely that Ipl1 and Mps1 communicate with each other via common substrates, or these two kinases have evolved to share the target residues because their substrates have physiologically related functions. This common feature of Ndc80 and Rad52 suggests that Rad52 works with Ipl1 and Mps1 in the same way as Ndc80 in the regulation of mitosis.

The various influences of Ipl1 on Mps1 have been well established. It has been reported that the kinase activity of Ipl1 is required for Mps1-induced SAC activation (46, 47) and Ipl1 generates unattached kinetochores to facilitate Mps1 binding to the kinetochores (48, 49). In addition to previous reports, we found evidence that the Ipl1 activity is crucial for not only the generation of unattached kinetochores but also efficient recovery of spindle damage, which is managed by Mps1-induced SAC (SI Appendix, Fig. S9A). Consistent with this result, Biggins and Murray (46) also observed that SAC activation by overexpression of Mps1 is not sufficiently maintained under suppression of Ipl1 activity. Taken together with our other data (Figs. 3B and 5A and SI Appendix, Fig. S7A), we conclude that Ipl1-dependent phosphorylation of Rad52 is required for the maintenance of high SAC activity during prolonged spindle damage conditions rather than for immediate response of the SAC to spindle damage. Given that Mps1 cycles rapidly through unattached kinetochores (50), it is likely that efficient and continuous recruitment of Mps1 to unattached kinetochores is important to maintain sufficient SAC activity. Thus, the defect in efficient accumulation of Mps1 at the kinetochores, which is caused by non–Ipl1-phosphorylatable mutation of Rad52, can lead to improper termination of the SAC under spindle damage conditions.

Contrary to these findings, Tanaka and colleagues reported that the localization of Mps1 is not affected by the suppression of Ipl1 activity (2). To understand the reasons for these conflicting results, we examined the effect of Ipl1 activity on the dynamics of Mps1 using a temperature-sensitive ipl1-321 mutant (39). Unfortunately, the Mps1 signal at the kinetochores during normal cell cycle progression was too weak to be detected (SI Appendix, Figs. S8A and S9B) and we could not examine the effect of Ipl1 activity on the localization of Mps1 during normal cell cycle progression. When the accumulation of Mps1 was induced by nocodazole treatment, ∼50% of cells showed the Mps1 signal at the kinetochores. Remarkably, the ratio of cells with the Mps1 signal at the kinetochores was decreased to ∼18% by the suppression of Ipl1 activity (SI Appendix, Fig. S9B). This observation is consistent with the results from using cells expressing Rad52[S374A], which is a non–Ipl1-phosphorylatable mutant of Rad52 (Fig. 6 B and C), and suggests that, although Mps1 localization to the kinetochores is not totally dependent on kinase activity of Ipl1, it is required for efficient localization of Mps1 to the kinetochores. A previous study using human cells provides an interesting explanation about the conflicting results for the dynamics of Mps1. According to Dou et al. (51), Mps1 binding to Ndc80 is dependent on two different domains and is regulated in an Aurora B (human homolog of Ipl1)-dependent or -independent manner. In prophase, inactive Mps1 binds to Ndc80 in an Aurora B-independent manner. In the following prometaphase, Mps1 is activated and more accumulated at the kinetochores in an Aurora B-dependent manner. Because the results from Tanaka and colleagues (2) were obtained during cell cycle progression after release from α-factor arrest, it is possible that Mps1 can localize to the kinetochores in an Ipl1-independent manner in prophase. Consistent with this possibility, the data from the Tanaka and colleagues (2) show Mps1 localization to the kinetochores before the duplication of spindle pole, indicating that cells were in prophase. Contrary to this, our results were obtained in prometaphase and metaphase. Thus, it is likely that the conflicting results for the dynamics of Mps1 may reflect two different manners of Mps1 binding to Ndc80, namely Ipl1-independent binding in prophase and Ipl1-dependent binding in prometaphase.

Despite the fact that the precise accumulation of Mps1 at the kinetochores is affected by Ipl1-dependent phosphorylation of Rad52, we observed that Mps1 still partially accumulates at the kinetochores in rad52-5A and even in rad52Δ cells (Fig. 6 B and C). This observation indicates that, although the regulation of Mps1 is largely dependent on Rad52, Rad52 is not the only factor responsible for Mps1 binding to the kinetochores. Given that the outer kinetochore subunit Ndc80 has been reported as an Mps1-binding protein in yeast and human cells (9, 45, 52), it is likely that Mps1, which is recruited by Rad52, tightly binds to the kinetochores via physical interaction with Ndc80. Furthermore, contrary to Rad52, a non-Ipl1/Aurora-phosphorylatable mutant of Ndc80 does not affect the accumulation of Mps1 at the kinetochores (53) and properly regulates the SAC under spindle damage conditions (44). These results strongly suggest that, similarly to Rad52, Ndc80 is also not the sole Ipl1 target for proper mitotic regulation and SAC regulation. Supposedly, Ndc80 may act as a direct binding platform for Mps1 localization, while Rad52 may act as a major Ipl1-dependent regulatory factor for Mps1 accumulation at the kinetochores. Given this, it is not surprising that deletion of RAD52 does not result in a total loss of kinetochore-binding affinity of Mps1.

Recently, it was reported that the DNA damage pathway-dependent regulation of Cep3 stimulates the SAC (54) and human Rad52 prevents force-induced DNA strand melting (55). In addition, some studies verified the involvement of components of the DNA damage pathway in the regulation of mitosis (35, 56). Although the detailed mechanisms are not completely understood, it is evident that there exists a crosstalk between the DNA damage pathway and the regulation of mitosis. In this study, we demonstrate that Rad52 function is not limited to DNA damage repair by homologous recombination activity. Through its ability to recruit Mps1 to the kinetochores and maintain SAC activity, Rad52 is a vital component of chromosome biorientation and is important for spindle damage repair by the SAC. In conclusion, it seems that Rad52 is a regulatory component of chromosome segregation in yeast mitosis. It would be interesting to confirm and extend the present findings in other eukaryotic cells.

Materials and Methods

Additional details for all procedures are in SI Appendix, SI Materials and Methods.

Fluorescence Microscopy.

Fluorescence microscopy was performed on a Nikon Eclipse Ti inverted microscope and a DeltaVision microscope (Applied Precision). Image analysis was performed using the NIS-Elements AR3.1 microscopy software (Nikon).

Vector Loss Assay.

Vector loss assay was performed using pRS415 vector as described previously (24).

ChIP Assay.

ChIP assay was performed as previously described (57). The primers used in the ChIP assay for CEN3 were 5′-ATCAGCGCCAAACAATATGG-3′ and 5′-AAAACTTCCACCAGTAAACG-3′, and those for noncentromeric DNA (CUP1) were 5′-TCTTTTCCGCTGAACCGTTCCAGC-3′ and 5′-GGCATTGGCACTCATGACCTTCAT-3′.

Measurement of DNA Content.

Flow cytometry was used to measure DNA content as previously described (58).

Western Blot Assay.

Western blot assay was performed by standard procedures. Each Western blotting experiment was performed at least three independent times.

In Vitro Kinase Assay.

In vitro kinase assay was performed as described previously (36) using Ipl1 and Rad52 variants purified from Escherichia coli by GST affinity purification and Mps1 purified from S. cerevisiae by immunoprecipitation.