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Vol. 17, Issue 6, 2746-2756, June 2006
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Department of Biomedical Sciences, College of Medicine and *Department of Biology, Florida State University, Tallahassee, FL 32306
Submitted December 1, 2005;
Revised February 13, 2006;
Accepted March 8, 2006
Monitoring Editor: Mark Solomon
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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hsl1, hsl7, and cdc5-2 mutants, which have defects in Swe1 degradation, show HU sensitivity because of high Swe1 protein levels. We further demonstrated that their HU sensitivity is not a result of DNA damage accumulation or incomplete DNA synthesis; instead the sensitivity is due to their dramatically delayed recovery from HU-induced S-phase arrest. Strikingly, our in vivo data indicate that Swe1 inhibits the kinase activity of Clb2-Cdk1, but not that of Clb5-Cdk1. Therefore, S-phase accumulated Swe1 prevents Clb2-Cdk1–mediated mitotic activities, but has little effects on Clb5-Cdk1–associated S-phase progression. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). However, Clb5 seems to play a more important role in DNA replication, as clb5 deletion mutants, but not clb6, results in a lengthened S-phase. It has been shown that Clb5 is responsible for both early and late origin firing, whereas Clb6 is only for early origin firing (Donaldson et al., 1998).
B-type cyclin-associated Cdk1 has been shown to phosphorylate a number of DNA replication factors, including components of the ORC, Orc1, Orc2, and Orc6, as well as Cdc6 and MCM proteins (Ubersax et al., 2003). One potential regulatory role for these phosphorylation events is to prevent reinitiation, thus restricting replication to a single round per cell cycle (Dahmann et al., 1995; Cocker et al., 1996; Piatti et al., 1996). Three other DNA replication–related proteins, Sld2/Drc1, Pol12, and Dpb2, have been shown to be the substrates of Clb-Cdk1 (Foiani et al., 1995; Masumoto et al., 2002; Kesti et al., 2004). Cdk1-dependent phosphorylation of Sld2 is the only one event known to be essential for the initiation of DNA replication in yeast. Phosphorylated Sld2 forms a complex with Dpb11 and is required for the loading of DNA polymerase 
to the origins (Masumoto et al., 2002). Pol12 is the second largest subunit of Pol. Its phosphorylation is cell cycle regulated and has been shown to promote the dissociation of Pol from the chromatin (Desdouets et al., 1998). Dpb2, the regulatory subunit of budding yeast Pol
, is also a substrate of Clb-Cdk1. Its phosphorylation is cell cycle regulated and believed to promote DNA synthesis, as mutation of the Cdk1 phosphorylation sites results in a synthetic phenotype with pol2-11 mutants, which show defects in DNA synthesis (Kesti et al., 2004).
Accurate DNA replication is critical to maintain the genetic stability. During S-phase, many intrinsic and extrinsic agents can affect DNA replication. S-phase checkpoint senses interrupted DNA replication, arrests cells at S-phase, inhibits late origin firing, and stabilizes replication forks (Lopes et al., 2001). It is still unclear how cell cycle machinery responds to interrupted DNA synthesis. Evidence from fission yeast, Xenopus, and mammals indicate that Wee1, a protein kinase that phosphorylates Cdk1 and inhibits its activity, is stabilized in response to DNA damage or DNA synthesis block (O'Connell et al., 1997; Michael and Newport, 1998; Raleigh and O'Connell, 2000). Moreover, recent evidence from vertebrate cells indicates that the destruction of Wee1 is essential for the recovery from DNA damage or S-phase checkpoint arrest. Plk1 is required for the recovery process because of its role in Wee1 protein degradation (van Vugt et al., 2004; Yamada et al., 2004). In budding yeast, the Wee1 homologue, Swe1, phosphorylates tyrosine 19 of Cdc28 and inhibits Cdk1 activity. However, neither swe1 deletion, nor CDC28F19 mutant, which is resistant to the phosphorylation by Swe1, exhibits checkpoint defects in the presence of DNA damage or DNA synthesis block (Amon et al., 1992; Sorger and Murray, 1992). It has been thought that the phosphorylation of Cdc28 may not be a factor in DNA synthesis regulation. Therefore, the regulation of Swe1 in response to the treatment of genotoxic agents has never been fully investigated.
Here we first described that Swe1 accumulates in response to DNA synthesis block by hydroxyurea (HU), a drug that inhibits ribonucleotide reductase and depletes the dNTP pool. To our surprise, Swe1 accumulation is not dependent on S-phase checkpoint, as mec1-1 and rad53 null mutants exhibit elevated Swe1 protein levels as well as wild-type (WT) cells. hsl1 and hsl7 mutants, which have defects in Swe1 degradation (Ma et al., 1996; McMillan et al., 1999; Shulewitz et al., 1999), show Swe1-dependent HU sensitivity. Similarly, cdc5-2 mutant also shows Swe1-dependent HU sensitivity due to the failure of Swe1 protein degradation. We further demonstrated that their HU sensitivity is a result of slow recovery from S-phase arrest, but not the results of DNA damage accumulation or slowed DNA synthesis. The accumulation of Swe1 delays the Clb2-Cdk1–dependent Pol12 phosphorylation dramatically, but has little effect on the Clb5-Cdk1–dependent phosphorylation of Sld2/Drc1. Thus budding yeast cells accumulate Swe1 protein during S-phase in order to block Clb2-Cdk1 associated mitotic activities.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To arrest yeast cells at G1-phase, 5 µg/ml -factor was added into midlog cell cultures (OD600 = 0.4) and the cultures were incubated for 2.5 h. To release them into cell cycle, the cell cultures were centrifuged and washed once with H2O. Nocodazole was purchased from ICN (Costa Mesa, CA) and used at 20 µg/ml in a final concentration of 1% dimethylsulfoxide.
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). Briefly, cells were fixed by the addition of 3.7% formaldehyde to growing cultures for at least 1 h. Cells were washed in phosphate-buffered saline, and microtubules were immunostained using the rat anti-tubulin antibody YOL1/34 and a FITC-conjugated secondary anti-rat antibody. DAPI was used to visualize DNA. The spindle and nuclei structures were visualized by fluorescence microscopy (Zeiss, Thornwood, NY).
Protein Techniques
Two milliliters of cell culture were used to prepare protein samples for time course experiments. Cells were collected in tubes with screw caps after being centrifuged and 50 µl of 20% TCA and glass beads were added. Cells were broken with a bead-beater for 2 min. Protein was precipitated by centrifugation at 3000 rpm for 2 min after glass beads were removed. Equal volume (50 µl) of 1 M Tris base and protein loading buffer were added. Dissolved protein samples were boiled for 5 min. Protein samples were resolved by 8% SDS-polyacrylamide gel electrophoresis (PAGE). Primary antibodies (anti-myc, anti-HA) were purchased from Covance (Madison, WI), and anti-Pgk1 antibody was from Molecular Probes (Eugene, OR). Anti-Pol12 was kindly provided by the Foiani lab in Italy. HRP-conjugated secondary antibody was purchased from Jackson ImmunoResearch (West Grove, PA).
Pulsed Field Gel Electrophoresis Analysis
Collected yeast cells were washed once with water and then fixed with 70% ethanol for 1 h at room temperature. To remove cell walls, cells were resuspended in LiSorb buffer and treated with zymolyase at 37°C for 1 h. Then cells were resuspended in TE buffer (10 mM Tris, 10 mM EDTA, pH 8.0). An equal volume of 1% melted agarose (after cooling down to 50°C) was added into cells. After agarose was solidified into agarose block, cells embedded in agarose blocks were subject to digestion with lysis buffer (100 mM EDTA, 10 mM Tris, 1% Sarkosyl, 100 µg/ml proteinase K, pH 8.0) overnight at 50°C. After that, agarose blocks were washed with TE buffer twice and were ready for pulsed field gel electrophoresis (PFGE) analysis. CHEF-DR II pulsed field electrophoresis systems (Bio-Rad, Richmond, CA) was used. The running time was 20 h at 6 V/cm with a 60-120-s switch time ramp (14°C).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). HU, an inhibitor of ribonucleotide reductase, is able to inhibit DNA synthesis and activate the S-phase checkpoint that arrests cells in S-phase. Recent evidence indicates that yeast cells exhibit Swe1-dependent hyphal growth when incubated on YPD plates containing HU (Jiang and Kang, 2003). In agreement with the previous data, we found that yeast cells also exhibited elongated cell morphology when incubated in liquid YPD medium containing 200 mM HU. Consistently, this morphology is also Swe1-dependent as swe1 deletion mutants did not show this phenotype (unpublished data). A reasonable explanation for these observations is that the presence of Swe1 protein in S-phase–arrested cells leads to hyphal growth. If that is the case, we expect to see high Swe1 protein levels in S-phase–arrested cells. Therefore, we examined Swe1 protein levels in the presence of HU. G1-arrested cells with myc-tagged SWE1 were released into YPD medium with different concentrations of HU at 30°C. After release, 20 µg/ml nocodazole was added into the medium to block the cells at metaphase when Swe1 protein levels are normally low. As shown in Figure 1A, Swe1 appeared at 40 and 60 min and then disappeared in the absence of HU. In the presence of HU, Swe1 appeared at the same time but kept persistent for a much longer time. The presence of either 50 or 200 mM HU resulted in Swe1 accumulation, but Swe1 protein accumulation is more dramatic in the presence of 200 mM HU. This result supports the conclusion that HU-induced hyphal growth is a result of Swe1 protein accumulation.
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rad53 sml1-1 SWE1-MYC strains were used for this experiment. The cells were arrested in G1-phase and then released into YPD medium containing 200 mM HU and 20 µg/ml nocodazole and incubated at 30°C. As shown in Figure 1B, Swe1 protein appeared in both WT and rad53 at 40 min. Strikingly, both of them maintained high Swe1 protein levels even 240 min after G1 release, suggesting that Swe1 accumulates in an S-phase checkpoint–independent manner. Consistently, both WT and rad53 cells showed hyphal growth phenotype after incubation in the presence of HU (Figure 1C). Because high levels of Swe1 protein induces hyphal growth, this result further supports the conclusion that Swe1 protein accumulation in the presence of HU does not depend on the S-phase checkpoint.
Hsl1/Hsl7 Complex Is Active in the Presence of HU
Bud neck–localized Hsl1/Hsl7 complex is responsible for bud neck recruitment and degradation of Swe1 (McMillan et al., 1999). Hsl1 is a protein kinase that phosphorylates Hsl7 and facilitates its bud neck localization (Shulewitz et al., 1999; Cid et al., 2001). Hsl7 binds to both Hsl1 and Swe1; thus, Hsl7 is thought to function as an adaptor. Recent evidence showed that the formation of the Hsl1 and Hsl7 platform at the bud neck is critical for Swe1 degradation (Asano et al., 2005). We have shown that Swe1 accumulates in the presence of HU. One possibility is that HU-induced Swe1 accumulation is attributed to Hsl1/Hsl7 inactivation. Therefore the localization of Hsl7 was examined in HU-arrested cells. WT cells with an HSL7-GFP plasmid were released into YPD medium containing 200 mM HU for 2 h, and Hsl7 localization was examined by fluorescence microscopy. As shown in Figure 2A, almost all the cells exhibited bud neck localized Hsl7 after S-phase arrest by HU. Because the bud neck localization of Hsl7 requires functional Hsl1, we conclude that Swe1 accumulation in the presence of HU is not a result of inactivation of the Hsl1/Hsl7 complex.
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). Because treatment of yeast cells with HU induces Cdc28 phosphorylation (Krishnan et al., 2004), it is possible that the inhibition of Clb2-Cdk1 after HU treatment results in Swe1 accumulation. Because Swe1 kinase phosphorylates Cdc28 at tyrosine 19, the CDC28F19 mutant, in which tyrosine 19 was substituted by phenylalanine, is resistant to the inhibition by Swe1 (Amon et al., 1992). If the inhibition of Cdc28 contributes to elevated Swe1 protein levels, CDC28F19 mutation should abolish HU-induced Swe1 accumulation. Thus Swe1 protein levels were compared in WT and CDC28F19 mutants. G1-arrested SWE1-MYC and CDC28F19 SWE1-MYC were released into YPD medium containing 200 mM HU and 20 µg/ml nocodazole. As shown in Figure 2B, Swe1 protein levels were persistent in both WT and CDC28F19 cells, although CDC28F19 mutant cells exhibited a little less Swe1 after a 120-min release into HU. This observation indicates that HU-induced Swe1 accumulation is not due to down-regulation of Clb2/Cdk1. Even though active Cdc28 is essential for Swe1 degradation, it is not sufficient to induce Swe1 degradation. Thus other factors essential for Swe1 degradation must be inhibited in HU-arrested yeast cells.
Mutants That Accumulate Swe1 Show HU Sensitivity
Hsl1, Hsl7, Cla4, and Cdc55 have been demonstrated to be involved in Swe1 protein degradation and deletion mutants of these genes exhibit Swe1-dependent hyphal growth. Bud-neck–localized Hsl1 and Hsl7 are able to facilitate the bud neck localization and degradation of Swe1 (McMillan et al., 1999; Shulewitz et al., 1999). Cla4 is a kinase that phosphorylates and promotes Swe1 degradation (Longtine et al., 2000). In addition, the Swe1 protein level has been shown to be elevated in the cdc55 mutant (Yang et al., 2000). If the elongated bud morphology in the presence of HU is a result of down-regulation of Hsl1, Hsl7, Cla4, or Cdc55, mutants of these genes will exhibit similar bud morphology in the presence or absence of HU. The hyphal growth of the four mutants was examined after incubation in the presence of 200 mM HU. Interestingly, all four mutants showed more pronounced hyphal growth in the presence of HU (Figure 3B and unpublished data), suggesting that the HU treatment and the mutation in the four genes have additive effects on the hyphal growth phenotype.
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hsl1, hsl7, cla4, and cdc55 mutants, these mutants may exhibit HU sensitivity. Indeed, we found that all four mutants showed HU sensitivity because they failed to form colonies on YPD plates containing 100 mM HU (Figure 3A). Because the four mutants exhibit defects in Swe1 protein degradation, it is likely that their HU sensitivity is a result of Swe1 accumulation. If this is the case, deletion of SWE1 should suppress their HU sensitivity. Therefore hsl1 swe1, hsl7 swe1, cdc55 swe1, and cla4 swe1 double mutants were constructed, and their HU sensitivity was examined on YPD plates containing 100 mM HU. Surprisingly, deletion of SWE1 only suppressed the HU sensitivity of hsl1 and hsl7 mutants; cdc55 swe1 and cla4 swe1 exhibited HU sensitivity similar to that of the single mutants (Figure 3C and unpublished data). However, the hyphal growth phenotype of the four mutants was all suppressed by SWE1 deletion, indicating that other Swe1-independent defects in cdc55 and cla4 mutants also contribute to HU sensitivity. Because Swe1 inhibits Cdc28 activity by phosphorylating a highly conserved tyrosine residue (Y19) at the N-terminus (Booher et al., 1993), we also tested whether the HU sensitivity of the hsl1 mutant depends on the phosphorylation of Cdc28 tyrosine 19. For this purpose, we examined the HU sensitivity of hsl1 CDC28F19, in which tyrosine 19 was substituted by phenylalanine. We found that CDC28F19 also suppressed the HU sensitivity of hsl1, suggesting that the inactivation of Cdk1 activity by elevated Swe1 protein leads to the HU sensitivity of the hsl1 mutant (Figure 3C).
cdc5-2 Mutant Shows Swe1-dependent HU Sensitivity
Cdc5, together with Cdk1 and Cla4, is involved in Swe1 phosphorylation and degradation (Sakchaisri et al., 2004). Because we have shown that hsl1 mutants exhibit HU sensitivity because of the failure of Swe1 degradation, cdc5 mutants defective in Swe1 degradation should also exhibit HU sensitivity. Thus, the HU sensitivity of two cdc5 mutant alleles was examined at room temperature, and cdc5-2 but not cdc5-1 exhibited HU sensitivity (Figure 4A and unpublished data). We reasoned that the HU sensitivity of cdc5-2 mutants should be a result of Swe1 accumulation, and this idea was tested by analyzing the HU sensitivity of cdc5-2 swe1 double mutants. As shown in Figure 4A, the swe1 null mutation completely suppressed the HU sensitivity of cdc5-2 mutants. The differential HU sensitivity of cdc5-1 and cdc5-2 mutants could reflect their distinct defects in Swe1 degradation. It is likely that the cdc5-2 mutant, but not cdc5-1, exhibits compromised Swe1 degradation.
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40 min and disappeared after 100 min in WT cells. In cdc5-2 mutants, Swe1 protein existed even after release for 160 min. Moreover, even G1-arrested cdc5-2 mutants exhibited low levels of Swe1 protein, indicating the defects of Swe1 degradation (Figure 4B).
Swe1 degradation is one of the essential functions of Cdc5 kinase (Asano et al., 2005). Because the cdc5-2 mutant accumulates Swe1 protein, the temperature-sensitive phenotype of cdc5-2 mutants could result from defects in Swe1 degradation. If this is true, deletion of SWE1 would suppress or partially suppress the temperature sensitivity of cdc5-2 mutant. Thus the growth of cdc5-2 and cdc5-2 swe1 mutants was examined at various temperatures. As expected, cdc5-2 swe1 double mutants grew much better than cdc5-2 single mutants at 30 or 33°C, the semipermissive temperature for cdc5-2. However, cdc5-2 swe1 failed to grow at 36°C, indicating that the cdc5-2 allele has other defects in addition to Swe1 protein degradation (Figure 4C). Indeed, we have previously demonstrated that cdc5-2 mutants exhibited defects in Bfa1 phosphorylation, which is required for mitotic exit (Hu et al., 2001).
Both Hsl1 and Cdc5 are required for Swe1 protein degradation, and hsl1 and cdc5-2 mutants exhibit HU sensitivity. Hsl1 and Cdc5 may function in a single pathway to control Swe1 degradation, or they regulate Swe1 protein levels in parallel pathways. To distinguish these possibilities, the hsl1 cdc5-2 double mutant was constructed, and its growth and HU sensitivity were examined. The double mutant displayed poor growth phenotype even when incubated at 25°C, and it was more HU sensitive than each single mutant (Figure 4D). Moreover, the sickness and the HU sensitivity of hsl1 cdc5-2 double mutants were suppressed by deleting SWE1 gene. Because hsl1 cdc5-2 double mutants exhibit a more severe phenotype than the single mutants, it is likely that Hsl1 and Cdc5 act in different pathways to control Swe1 protein degradation.
Swe1 Accumulation in hsl1 Mutant Causes a Slow Recovery from S-phase Arrest
Next we asked why hsl1 and hsl7 mutants show Swe1-dependent HU sensitivity. One possibility is that hsl1 and hsl7 mutants fail to arrest the cell cycle in the presence of HU. To test this, we examined the spindle morphology of WT and hsl1 mutant cells in the presence of 200 mM HU. G1-synchronized WT and hsl1 mutant cells were released into YPD medium containing 200 mM HU and incubated at 30°C. The spindle staining of the collected cells revealed that both WT and hsl1 mutant cells exhibited short spindle structure even after incubation for 3 h (unpublished data). The typical S-phase spindle structures in hsl1 mutant cells in the presence of HU suggests the intact S-phase checkpoint function (Alcasabas et al., 2001). Moreover, no obvious loss of viability was observed for hsl1 mutant cells after incubation in the presence of 200 mM HU for 6 h.
As our result indicates no checkpoint defects in hsl1 mutants, we then tested the possibility that persistent Swe1 protein levels prevent cell cycle progression after the removal of HU. Recent data indicate that the degradation of Wee1 protein, the Swe1 homologue in higher eukaryotes, is required to resume cell cycle progression after removal of genotoxic agents (van Vugt et al., 2004; Yamada et al., 2004). It is possible that budding yeast shares the same regulation. To test this possibility, G1-arrested WT, hsl1, and hsl1 swe1 mutant cells were released into YPD medium containing 200 mM HU. After incubation in the presence of HU for 100 min, HU was washed off and the cells were resuspended in YPD medium. -factor was then added into the medium to block the second round of the cell cycle. Cells were collected and fixed for spindle staining and budding index. In the presence of HU, WT, hsl1, and hsl1 swe1 were arrested in S-phase with short spindle structures (Figure 5A). Sixty minutes after HU removal, the short spindle structure disappeared in majority of WT cells, suggesting that cells entered anaphase. However, it took hsl1 mutant cells much longer to enter anaphase after HU removal. One hundred minutes after HU removal, more than 40% of hsl1 mutant cells still exhibited short spindle structure. Strikingly, swe1 deletion completely suppressed the delay of anaphase entry in hsl1 mutants after HU removal (Figure 5A), indicating that Swe1-dependent delay of cell cycle resumption after HU removal contributes to the HU sensitivity of hsl1 and hsl7 mutants.
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hsl1 mutants correlates with Swe1 accumulation, Swe1 protein levels were examined in WT and hsl1 mutants using a protocol similar to the one mentioned above. Briefly, G1-synchronized cells were released into YPD medium containing 200 mM HU for 100 min. After HU was washed off, the cells were released into YPD medium containing 20 µg/ml nocodazole to block the cell cycle at metaphase when Swe1 protein levels are low. In WT cells, Swe1 protein disappeared 60 min after HU removal, consistent with the timing of anaphase entry. However, Swe1 protein levels were persistent in hsl1 mutant cells even at the 200-min time point when HU was removed for 100 min (Figure 5B). Thus hsl1 mutant exhibits dramatically delayed disappearance of Swe1 after HU treatment compared with WT cells. This observation suggests that the accumulation of Swe1 protein in the hsl1 mutant is responsible for slower recovery from HU-induced S-phase arrest.
The Slow Recovery in the hsl1 Mutant Is Not a Result of Accumulated DNA Damage
It has been shown that deletion of CLB5 is lethal in the absence of RAD53 due to the accumulated DNA damage in clb5 mutants (Gibson et al., 2004). This observation raises a possibility that the slow recovery in hsl1 mutants could be a result of accumulation of damaged DNA due to the inhibition of Clb5-Cdk1 by Swe1. If that is the case, damaged DNA in hsl1 mutants should activate the DNA damage checkpoint pathway and delay recovery from S-phase arrest. Thus the existence of damaged DNA was examined in hsl1 mutants by analyzing the phosphorylation of Rad53, a key player in both S-phase and DNA damage checkpoint pathways. Rad53 is phosphorylated by Mec1 kinase in response to DNA damage or incomplete DNA synthesis (Sanchez et al., 1996). The phosphorylation of Rad53 has been widely used as a marker for checkpoint activation. RAD53-HA and hsl1 RAD53-HA strains were synchronized at G1-phase and then released into YPD medium containing 200 mM HU. One hundred minutes later, HU was washed off and the cells were resuspended in YPD medium containing nocodazole as described earlier. Protein samples were prepared to examine the phosphorylation of Rad53. As shown in Figure 5A, both WT and hsl1 mutant cells exhibited phosphorylated Rad53-HA in the presence of HU, as judged by a protein band shift. After the removal of HU, the phosphorylated Rad53 protein disappeared in 60 min in WT cells. Both WT and hsl1 mutant cells exhibited similar kinetics for the disappearance of phosphorylated Rad53 (Figure 6A). Thus the slow recovery phenotype in hsl1 mutants is not due to the presence of DNA damage.
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hsl1 Mutants Is Not a Result of Delayed DNA Synthesishsl1 mutants after exposure to HU could be a result of slowed DNA synthesis due to the high levels of Swe1. If so, we expect to observe delayed completion of DNA synthesis in hsl1 mutants after they are challenged by HU. Completion of chromosome replication allows chromosomes to be resolved by PFGE. Before completion of DNA replication, chromosomes contain structures such as replication forks and bubbles that prevent migration of chromosomal DNA into the gel (Desany et al., 1998; Schollaert et al., 2004). Thus we used PFGE to follow the completion of DNA synthesis in WT and hsl1 mutant cells after HU treatment.
Cells synchronized in G1 were released into the cell cycle in medium containing 200 mM HU. After 100 min, the cells were washed and resuspended in medium containing nocodazole as described earlier. The chromosomes from cells collected during and after HU treatment were separated by PFGE. As shown in Figure 6B, WT cells recovered from transient exposure to 200 mM HU and completed DNA replication, as evidenced by the appearance of the chromosomes signal after 160 min. Surprisingly hsl1 mutant cells completed DNA replication at the same time (160 min) under these conditions (Figure 6B). Therefore, the delayed recovery in hsl1 mutants is not a result of slow DNA synthesis. In another word, Swe1 accumulation does not affect DNA synthesis. Indeed, we have found that overexpression of Swe1 results in the cell cycle arrest and hyphal growth, but the S-phase progression was not affected by high dosage of Swe1 based on the FACS analysis (unpublished data).
Clb2-Cdk1 Might Be Down-regulated in hsl1 and hsl7 Mutants
If the failure of the growth of hsl1 and hsl7 on HU plates is the result of inhibition of Clb-Cdk1, overexpression of B-type cyclins may suppress the HU sensitivity of hsl1 and hsl7 mutants. Thus a PGAL-CLB5, PGAL-CLB2, and a control vector were transformed into hsl7 mutants, and the growth of the transformants was examined on URA dropout plates containing galactose and 100 mM HU. Overexpression of either CLB5 or CLB2 suppressed the HU sensitivity of hsl7 mutants, but overexpression of CLB2 exhibited a more dramatic suppression phenotype (Figure 7A). This result indicates that Clb2-Cdk1 might be the physiological target of Swe1 and overproduction of Clb2 reverses the inhibition of Clb2-Cdk1 by Swe1.
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; Kellogg and Murray, 1995). Thus we speculate that Swe1-dependent hyphal growth in the presence of HU should be a result of inhibition of Clb-Cdk1. If that is the case, deletion of B-type cyclins should promote hyphal growth in the presence of HU. Therefore we examined the cell morphology of all the B-type cyclin deletion mutants (from ATCC, Manassas, VA) in the presence of 200 mM HU. Among all the B-type cyclin deletion mutants (clb1, clb2, clb3, clb4, clb5, and clb6), only the clb2 mutant exhibited dramatic hyphal growth (Figure 7B). This observation suggests that the inhibition of Clb2-Cdk1, but not other B-type cyclin-associated Cdk1, contributes to the hyphal growth phenotype in the presence of HU.
Clb5-Cdk and Clb2-Cdk1 Exhibit Differential Sensitivity to Swe1
It is well established that Swe1 phosphorylates Cdk1 and inhibits its kinase activity, but our observation indicates that accumulated Swe1 does not affect DNA synthesis (Figure 6). Moreover, overexpression of CLB2 suppresses the HU sensitivity of hsl7 better than CLB5. Clb5 and Clb6 are S-phase cyclins and Clb5-Cdk1 plays a major role in DNA synthesis. One obvious question is whether S-phase cyclin-conjugated Cdk1 is inhibited by accumulated Swe1 during S-phase. Recently, the Morgan laboratory compared the specificity of the S-phase cyclin Clb5 and the M-phase cyclin Clb2 in the phosphorylation of 150 Cdk1 substrates. Some of these proteins were phosphorylated more efficiently by Clb5-Cdk1 than Clb2-Cdk1. Sld2 is one of the Clb5-specific targets and it is involved in early S-phase events (Loog and Morgan, 2005). Sld2/Drc1 is required for DNA replication and plays a role in checkpoint signaling of stalled replication forks (Wang and Elledge, 1999). Phosphorylation of Sld2 by Clb5-Cdk1 promotes its association with Dpb11 and the subsequent loading of Cdc45 and DNA polymerase (Masumoto et al., 2002). So we first examined if the phosphorylation of Sld2 is delayed in hsl1 mutants.
To examine the effect of Swe1 on Sld2 phosphorylation, G1-arrested WT and hsl1 mutant cells with 9myc-tagged SLD2 were released into YPD medium containing 50 mM HU and 20 µg/ml nocodazole at 25°C. Protein samples were prepared every 15 min, and Sld2 protein phosphorylation was examined after Western blot analysis. At 30 min after G1 release, both WT and hsl1 mutant cells exhibited a slow migrating band of Sld2, indicating that Sld2 is phosphorylated by Clb5-Cdk1 at this time. Clearly, there is no any delay in Sld2 phosphorylation in hsl1 mutants (Figure 8A). Because we have shown that Swe1 accumulates in the presence of 50 mM HU (Figure 1), it is very likely that Clb5-Cdk1 is not inhibited by the presence of Swe1.
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; Masumoto et al., 2002; Kesti et al., 2004). Like Sld2, Dpb2 is phosphorylated more efficiently by Clb5-Cdk1 than Clb2-Cdk1, whereas Pol12 is not a Clb5-Cdk1–specific target (Loog and Morgan, 2005). Because Clb2-Cdk1 possesses higher intrinsic kinase activity than Clb5-Cdk1 (Loog and Morgan, 2005), we speculate that Pol12 is more efficiently phosphorylated by Clb2-Cdk1. To determine if the phosphorylation of Pol12 and Dpb2 is inhibited by Swe1 accumulation, WT cells, hsl1, DBP2-myc, and hsl1 DPB2-myc were synchronized at G1-phase and then released into YPD medium containing 50 mM HU and 20 µg/ml nocodazole as described in Figure 8A. For WT cells, the phosphorylated Dpb2 appeared at 45 min, and the majority of Dpb2 exhibited as hyperphosphorylated forms at 105 min after G1 release (Figure 8B). In hsl1 mutants, phosphorylated Dpb2 did not appear until 60 min after G1 release and the majority of Dpb2 became phosphorylated 150 min after G1 release. Moreover, at 75 min, WT cells exhibited equal amount of hypo- and hyperphosphorylated Dpb2 protein, but it took 105 min for hsl1 mutants to show a similar ratio (Figure 8B). It is clear that the phosphorylation of Dpb2 by Cdk1 is delayed, but it could be phosphorylated eventually.
The kinetics of Pol12 phosphorylation was also examined in WT and hsl1 mutants with anti-Pol12 antibody (Piatti et al., 1996). In WT cells, the phosphorylated Pol12 appeared at 105 min after G1 release, much later than Sld2 and Dpb2 (Figure 8C). However, obvious phosphorylation of Pol12 was not observed even at the last time point in hsl1 mutants in the presence of 50 mM HU (180 min; Figure 8C), indicating that HU treatment blocks Pol12 phosphorylation completely. We then asked if the delayed Pol12 phosphorylation depends on Swe1 accumulation. For this purpose, swe1 and swe1 hsl1 mutant cells were synchronized at G1-phase and then released into YPD medium containing 50 mM HU and 20 µg/ml nocodazole at 25°C. As shown in Figure 8C, the phosphorylated Pol12 appeared at 75 min in swe1 mutant cells, 30 min earlier than WT cells. Moreover, we did not observed any difference between swe1 and swe1 hsl1 mutants regarding to the kinetics of Pol12 phosphorylation (Figure 8C). These observations indicate that both HU treatment and hsl1 deletion contribute to delayed Pol12 phosphorylation, and this delay is a result of Swe1 accumulation.
It is obvious that accumulated Swe1 protein delays the phosphorylation of some Cdk1 substrates, but different Cdk1 substrates exhibit a distinct response to the accumulation of Swe1. We noticed that the timing of the phosphorylation of Sld2, Dpb2, and Pol12 during the cell cycle was different. In the presence of 50 mM HU, WT cells showed phosphorylated Sld2, Dpb2, and Pol12 at 30, 45, and 120 min, respectively (Figure 8). It seems that proteins that are phosphorylated later in the cell cycle are more sensitive to Swe1 accumulation. Moreover, Sld2 and Dpb2 are two Clb5-Cdk1–specific substrates; thus the accumulation of Swe1 only inhibits Clb2-Cdk1, but not Clb5-Cdk1.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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hsl1 and hsl7 mutants, which are unable to destroy Swe1, exhibited HU sensitivity because of the failure of the resumption of cell cycle after HU treatment. We further demonstrated that the delayed recovery after HU treatment in hsl1 and hsl7 mutants is not a result of slowed DNA synthesis or DNA damage accumulation. Interestingly, the kinase activity of Clb2-Cdk1, but not Clb5-Cdk1, is inhibited in the presence of high levels of Swe1 protein. Therefore, the presence of Swe1 during S-phase inhibits Clb2-Cdk1–associated mitotic activities. On the other hand, the destruction of Swe1 after DNA synthesis is required to initiate mitosis.
In the presence of HU, activated S-phase checkpoint arrests cells at S-phase, inhibits late origin firing, and stabilizes replication forks (Osborn et al., 2002). We demonstrated that yeast cells treated with HU maintain high levels of Swe1. To our surprise, mec1-1 and rad53 mutants also exhibit persistent levels of Swe1 protein in the presence of HU, arguing against the possibility that the activated S-phase checkpoint leads to Swe1 accumulation. In contrary to previous data, in which HU-induced hyphal growth is suppressed by the S-phase checkpoint (Jiang and Kang, 2003), we found that rad53 deletion mutants exhibit obvious hyphal growth when incubated in liquid medium containing HU (Figure 1C), further supporting the notion that Swe1 accumulates in the absence of the S-phase checkpoint. The difference could be due to the different incubation conditions. We examined the morphology of cells grown in liquid YPD medium containing 200 mM HU, whereas the previous work was done with YPD plates containing 100 mM HU.
The degradation of Swe1 requires protein kinases Cdk1, Cdc5, and Cla4. Hsl1 and Hsl7 complex provides a platform at the bud neck for Swe1 degradation (Asano et al., 2005). It has been shown that the Swe1 protein level is elevated in cdc55 mutants, and this is consistent with the observation that cdc55 mutants exhibit Swe1-dependent hyphal growth (Yang et al., 2000). We found that hsl1, hsl7 mutants, but not cla4 and cdc55, showed Swe1-dependent HU sensitivity. Thus other defects than Swe1 degradation in cla4 and cdc55 mutants may also contribute to their HU sensitivity. Indeed, Cla4 is required for mitotic exit, and Cdc55 plays a role in mitotic exit and sister chromatid separation (Wang and Ng, 2006). Moreover, recent research already showed that the inhibition of PP2A impairs DNA repair and cells are hypersensitive to DNA damage (Chowdhury et al., 2005), suggesting that HU sensitivity of cdc55 may be due to defects in DNA repair. It will be interesting to understand the mechanism of Swe1-independent HU sensitivity of cla4 and cdc55 mutants.
Different from hsl1 and hsl7, cdc5-2 mutant exhibited Swe1-dependent HU sensitivity without any obvious hyphal growth phenotype (unpublished data). This observation indicates that Swe1-induced hyphal growth in hsl1 and hsl7 mutants is not the cause of their HU sensitivity. Instead, the elevated Swe1 protein levels in hsl1 or hsl7 mutants result in both HU sensitivity and hyphal growth. It is possible that the hyphal growth phenotype is suppressed in cdc5-2 mutants. For example, the defect of cdc5-2 in anaphase entry and mitotic exit may lead to higher Clb2 levels that inhibit hyphal growth (Alexandru et al., 2001; Hu et al., 2001).
It is not clear how HU-induced S-phase arrest leads to Swe1 accumulation. One possibility is that SWE1 is transcribed during S-phase, and HU arrests cells at S-phase, which contributes to consistent SWE1 transcription. Alternatively, the presence of HU may induce Swe1 accumulation through inhibiting Swe1 degradation machinery. HU-arrested S-phase cells exhibit bud-neck localized Hsl7 protein, indicating that the Hsl7 pathway is functional in the presence of HU. Moreover, the inactivation of Cdk1 may not be the reason for Swe1 accumulation, because the CDC28F19 mutant, which is resistant to the inhibitory phosphorylation of Swe1, also exhibits comparable Swe1 protein levels in the presence of HU. The inhibition of Cdc5 kinase activity in HU-treated cells could contribute to Swe1 stabilization, based on the following observations. First, cdc5-2 shows Swe1-dependent HU sensitivity, and Swe1 protein degradation is compromised in cdc5-2 mutants. Moreover, Cdc5 kinase activity is largely inhibited in the presence of HU because there is little phosphorylation of its substrate Bfa1 (Hu et al., 2001). The fact that treatment of Cdc5 protein with phosphatase abolishes its kinase activity indicates the presence of a protein kinase responsible for the phosphorylation and activation of Cdc5 in budding yeast (Cheng et al., 1998). Recent data from fission yeast indicate that a protein kinase phosphorylates Plo1, the yeast Cdc5 homologue, at Ser 402 and this phosphorylation is required for recovery after stress (Petersen and Hagan, 2005). Thus, to identify the protein kinase that phosphorylates Cdc5 will be the key to understand the regulation Swe1 protein levels in response to DNA synthesis block.
We have demonstrated that Swe1 degradation is required for the recovery from HU-induced S-phase arrest. Similarly, destruction of Wee1 has been shown to be required to resume the cell cycle in vertebrate cells after challenge by DNA damage or DNA synthesis interruption. Plk1, the Cdc5 homologue in mammals, is required for the recovery after DNA damage (van Vugt and Medema, 2004). In Xenopus, it has been demonstrated that Wee1 exhibits Hsl7-dependent accumulation after DNA synthesis inhibition by HU (Yamada et al., 2004). It remains to be tested if degradation of Wee1 is required for the recovery after DNA synthesis block in mammals. It is likely that all eukaryotic cells share a similar mechanism to recover from the cell cycle arrest after stress. This process might be achieved by activating Cdc5 kinase in budding yeast or its homologues in other eukaryotic organisms.
What is the biological significance of Swe1 accumulation in response to interrupted DNA synthesis? One possibility is that the presence of Swe1 in the presence of HU inhibits DNA synthesis. For example, Clb5-Cdk1 might be inhibited so that the later origin firing is blocked. But our observations argue against this model. First, the phosphorylation of Sld2/Drc1, one of the Clb5-Cdk1 specific substrate, exhibits similar kinetics in WT and hsl1 mutants, even though there is much more Swe1 protein in hsl1 mutants. Second, after exposed with HU, hsl1 mutants complete DNA synthesis without any obvious delay compared with WT cells, indicating that the inhibition of late origin firing after S-phase checkpoint activation in the presence of HU is not dependent on Swe1. Thus, the delayed DNA synthesis does not contribute to the slow recovery in hsl1 mutants. We also clarified that the slow recovery phenotype in hsl1 mutants is not a result of DNA damage accumulation.
We compared the phosphorylation kinetics of three Cdk1 substrates, Sld2/Drc1, Dpb2 and Pol12, in response to Swe1 accumulation. Surprisingly, the three Cdk1 substrates showed differential sensitivity to high Swe1 protein levels. As shown in Figure 8, the phosphorylation of Sld2/Drc1 does not show any sensitivity to Swe1 accumulation. In contrast, Pol12 exhibits the most dramatic sensitivity to Swe1 accumulation. As Sld2/Drc1 is a specific substrate of Clb5-Cdk1, whereas Pol12 is a Clb2-Cdk1 substrate, we therefore conclude that Clb2-Cdk1 is more sensitive to Swe1 than Clb5-Cdk1. In agreement with this, we found that deletion of CLB2, but not other B-type cyclins, results in dramatic hyphal growth in the presence of HU (Figure 7), indicating that Swe1 induced hyphal growth is due to the inhibition of Clb2-Cdk1. Recent published data from Aparicio's lab confirm this speculation. The in vitro kinase activity of Clb2-Cdk1 was inhibited dramatically in the presence Swe1, but Clb5-Cdk1 was unaffected (Hu and Aparicio, 2005). Thus, this conclusion can well explain the observation that high levels of Swe1 during S-phase do not affect Clb5-Cdk1–dependent S-phase progression.
Our results suggest that Swe1 prevents Clb2-associated mitotic activities during S-phase. If that is the case, deletion of SWE1 should exhibit premature mitotic activities in S-phase–blocked cells. We did observed earlier Pol12 phosphorylation in swe1 deletion mutants, indicating the premature activation of Clb2-Cdk1. However, the swe1 mutant does not show any obvious HU sensitivity. It is likely that the periodic expression of mitotic cyclins limits their activity during S-phase and makes Swe1 dispensable for S-phase regulation. Therefore, both Swe1 and lower transcription levels of CLB2 gene in S-phase may prevent any mitotic activities stimulated by Clb2-Cdk1. It is also possible that other unidentified pathways keep Clb2-Cdk1 inactive in S-phase–blocked cells.
In summary, we have identified a new type regulation in response to DNA synthesis block in budding yeast. Swe1 accumulates when DNA synthesis is interrupted, and degradation of Swe1 is required for the resumption of cell cycle after the removal of DNA replication stress. This accumulation may be a result of the inhibition of Cdc5 kinase activity. Accumulated Swe1 prevent Clb2-Cdk1 activation, but has no effect on S-phase cyclin-associated Cdk1 activity. This regulation is conserved from yeast to human cells, and studies on the regulation and function of Swe1 in response to interrupted DNA synthesis will help us understand how eukaryotic cells maintain the genome integrity when genotoxic agents are present.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Yanchang Wang ( yanchang.wang{at}med.fsu.edu)
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REFERENCES |
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