An Essential Role for the Saccharomyces cerevisiae DEAD-Box Helicase DHH1 in G1/S DNA-Damage Checkpoint Recovery

Corresponding author: Joseph C. Reese, Penn State University, 203 Althouse Lab, University Park, PA 16802., jcr8{at}psu.edu (E-mail)

Communicating editor: F. WINSTON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The eukaryotic cell cycle displays a degree of plasticity in its regulation; cell cycle progression can be transiently arrested in response to environmental stresses. While the signaling pathways leading to cell cycle arrest are beginning to be well understood, the regulation of the release from arrest has not been well characterized. Here we show that DHH1, encoding a DEAD-box RNA helicase orthologous to the human putative proto-oncogene p54/RCK, is important in release from DNA-damage-induced cell cycle arrest at the G1/S checkpoint. DHH1 mutants are not defective for DNA repair and recover normally from the G2/M and replication checkpoints, suggesting a specific function for Dhh1p in recovery from G1/S checkpoint arrest. Dhh1p has been suggested to play a role in partitioning mRNAs between translatable and nontranslatable pools, and our results implicate this modulation of mRNA metabolism in the recovery from G1/S cell cycle arrest following DNA damage. Furthermore, the high degree of conservation between DHH1 and its human ortholog suggests that this mechanism is conserved among all eukaryotes and potentially important in human disease.

THE G1-to-S phase transition, termed START in yeast, represents an important and thus highly regulated decision point in the cell cycle, as it signifies a commitment to completion of cell division (LEVINE et al. 1995 ; REED 1997 ). Eukaryotic cells are capable of undergoing a transient arrest at the G1/S transition if conditions that would be unfavorable for cell division, such as nutrient limitation (GALLEGO et al. 1997 ), environmental toxins (PHILPOTT et al. 1998 ), or damaged DNA are encountered. This capacity for transient arrest allows the cell to respond to environmental stresses in such a way that viability is maximized. Disruption of either the ability to initiate the arrest or the ability to subsequently recover from the arrest and resume cell division appears to be detrimental (HARTWELL et al. 1994 ; LYDALL and WEINERT 1995 ; SHAULIAN et al. 2000 ).

In the case of DNA damage, much more is known about the signaling cascade leading to the initiation of the transient arrest, known as the checkpoint response, than about the mechanisms regulating the subsequent release from checkpoint arrest. The DNA-damage signaling cascade appears to be highly conserved throughout eukaryotes (LYDALL and WEINERT 1996 ). Damage activates a series of phosphorylation events leading to phosphorylation of the ATM homologs, MEC1 and TEL1 (MORROW et al. 1995 ; SIEDE et al. 1996 ). These lipid kinase family members are believed to be partially redundant in the DNA-damage signaling cascade and cause phosphorylation of a kinase encoded by RAD53 (SANCHEZ et al. 1996 ; SUN et al. 1996 ). Rad53p then phosphorylates the transcription factor component Swi6p, which causes a delay in the accumulation of mRNA for G1 cyclins and thus a transient cell cycle arrest (SIDOROVA and BREEDEN 1997 ). While much is understood about the initiation of the transient G1/S checkpoint arrest, little is known about the downstream events, regulating release from the arrest. Our evidence suggests that the yeast gene DHH1 plays a role in this process.

DHH1 encodes a highly conserved putative DEAD-box RNA helicase that has been shown to associate with factors that are reported components of mRNA decapping, deadenylation, and transcription complexes in yeast (COLLER et al. 2001 ; FISCHER and WEIS 2002 ; MAILLET and COLLART 2002 ). Dhh1p stimulates mRNA decapping by the decapping enzyme Dcp1p, and it has been shown to localize, along with other proteins involved in decapping and mRNA degradation, to discrete cytoplasmic foci known as P-bodies. P-bodies are believed to be involved in sequestering mRNAs in a nontranslating pool, from which they are subsequently degraded or possibly reactivated for translation (SHETH and PARKER 2003 ). Highly conserved orthologs of Dhh1p have been shown to play a role in repressing translation of specific messages as part of the intricate program of translational control that operates in early development of Xenopus (SMILLIE and SOMMERVILLE 2002 ), Drosophila (NAKAMURA et al. 2001 ), and Spisula solidissima (clam; MINSHALL et al. 2001 ). The Xenopus ortholog of Dhh1p, Xp54, is known to localize to stored maternal mRNPs in oocytes (LADOMERY et al. 1997 ), where its role in sequestering mRNAs to a nontranslating pool in discrete cytoplasmic foci appears to be analogous to the role of Dhh1p in yeast P-bodies. Indeed, overexpression of Xp54 in a dhh1 yeast strain can functionally compensate for the lack of Dhh1p (TSENG-ROGENSKI et al. 2003 ).

The significance of the interactions between Dhh1p and components of deadenylation and transcription complexes is less clear. Physical and genetic interactions between Dhh1p and Ccr4p, Pop2p, and Not1p have been reported (HATA et al. 1998 ; COLLER et al. 2001 ; MAILLET and COLLART 2002 ). Ccr4p is the catalytic subunit in the yeast deadenylation machinery, and Pop2p and at least some of the Not proteins also appear to be components of the deadenylation complex (TUCKER et al. 2001 , TUCKER et al. 2002 ). The Ccr4-Not complex is also believed to act in the regulation of transcription (COLLART and STRUHL 1994 ; HATA et al. 1998 ; DELUEN et al. 2002 ). It has been suggested that the Ccr4-Pop2-Not complex may act to coordinate the behavior of a transcript from its transcription through its deadenylation (TUCKER et al. 2001 ; MAILLET and COLLART 2002 ), and the reported interactions of Dhh1p with components that appear to be involved in both regulating transcription (the Not proteins) and decapping (Dcp1p and Dcp2p) suggests Dhh1p may contribute to or even extend this coordinated regulation.

Interestingly, the human homolog of DHH1, p54/RCK (DDX6), is a target gene of a chromosomal translocation breakpoint (11q23.3) fusion from a B-cell lymphoma and is overexpressed in several malignant cell types (LU and YUNIS 1992 ; AKAO et al. 1995 ; NAKAGAWA et al. 1999 ); thus, it is a candidate proto-oncogene.

Here we show that dhh1 mutant cells are hypersensitive to DNA damage and deficient in cell cycle reentry following activation of the G1/S checkpoint by DNA damage. These defects are specific to DNA damage-induced G1/S arrest, as DHH1 is not required for recovery from -factor-induced G1 arrest or for recovery from the replication (S) or G2/M DNA-damage checkpoints. Partially inactivating the G1/S checkpoint by deletion of MEC1 can alleviate the requirement for DHH1 in passing through START following DNA damage. However, overriding cell cycle checkpoints completely or constitutively overexpressing the G1 cyclin CLN3 is lethal in dhh1 cells, even in the absence of exogenous DNA damage. Deleting the gene encoding the mRNA decapping enzyme Dcp1 likewise caused increased sensitivity to DNA damage. In conjunction with the recent studies that strongly implicate Dhh1p and its orthologs in regulation of mRNA stability and translation, these results suggest that crucial aspects of G1/S checkpoint regulation are carried out at the level of mRNA metabolism. The conservation of sequence and function between DHH1 and its human ortholog implies that the disruption of normal checkpoint functions is a likely mechanism for p54/RCK-associated oncogenesis.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains and growth conditions:
All strains are isogenic with PH499 (MATa, ade2-101 ochre his3-200 leu2-1 ura3-52 trp1-63 lys2-801 amber), unless otherwise noted. Strains used throughout these studies are listed in Table 1. Strains YRP840 (DCP1) and YRP1071 (dcp1) were described previously (THARUN and PARKER 2001 ). The coding sequence of p54/RCK (DDX6) was amplified by PCR from a HeLa cell cDNA expression library and cloned into a derivative of a yeast expression vector (pG1; SCHENA et al. 1991 ) containing two HA epitope sequences inserted at the BamHI site. CLN3 derivatives were expressed from the GAL1 promoter using the vector pYES2 (Clontech, Palo Alto, CA). The destruction box mutant (CLN3DB) was constructed by introducing a stop codon at residue 398. Cultures for fluorescence-activated cell sorting (FACS) analysis were grown in minimal media (yeast nitrogen base, dextrose, and complete amino acids). Other cultures were grown in rich media (1% yeast extract, 2% Bacto-Peptone, and 2% dextrose). Doses of ultraviolet (UV) radiation or methyl methanesulfonate (MMS) and growth conditions following treatment with DNA-damaging agents are described in the figure legends.

FACS and checkpoint analysis:
Synchronization in G1 was achieved by treatment of a midlog phase culture with -factor (Sigma, St. Louis) at a concentration of 0.2 µg/ml (or 5 µg/ml for strains with an intact BAR1 gene) for 3–3.5 hr. Synchronization in S phase was achieved by treatment of a midlog phase culture with hydroxyurea at a concentration of 150 mM for 3–3.5 hr, and G2/M synchronizaton was achieved by treatment of a midlog phase culture with nocodazole at a concentration of 10 µg/ml for 3–3.5 hr. To induce DNA damage by ultraviolet radiation, cultures were centrifuged and resuspended in a small volume of media, spread onto 150-mm solid media plates at a density of 13–14 OD_600nm units per plate, and then exposed to either 50 or 60 J/m² of UV light. Cells were then scraped off the plates, further washed to remove -factor, and resuspended in fresh media at OD_600nm of 0.5. To induce DNA damage by the alkylating agent MMS, cultures were treated with MMS at a concentration of 0.2% for 30 min. Cells were then collected by centrifugation, washed once with media containing 10% sodium thiosulfate to inactivate the MMS, and washed twice more with fresh media to remove -factor, before resuspension in fresh media at OD_600nm of 0.5. Cells were collected and RNA was isolated for Northern blotting or processed for FACS analysis and budding indices were measured as described in previous publications (WEINERT et al. 1994 ; SIDOROVA and BREEDEN 1997 ).

DNA repair assay:
DNA-damage repair assays were conducted as described in a previous publication with modifications (GILLETTE et al. 2001 ).

DHH1 (YJR218), dhh1 (YJR219), and rad23 (Research Genetics, Birmingham, AL) cells were grown in YPAD at 30° to an OD₆₀₀ of 0.6–0.7. A 50-ml aliquot of cells was collected by centrifugation, washed in sterile water, and resuspended into 40 ml ice-cold PBS. From this point on all manipulations were performed under low-light conditions and using amber centrifuge tubes. Twenty milliliters of the cell suspension was transferred to each of two 150-mm petri dishes and exposed to 40–60 J/m² UV irradiation using a Stratalinker 2400 (Stratagene, La Jolla, CA). The cells in the experiment shown in Fig 4 were exposed to 50 J/m², but dhh1 cells repaired DNA damage as well as the wild type when exposed to 40, 50, or 60 J/m² of UV irradiation (not shown). The cells were transferred to a 50-ml tube and an aliquot was immediately removed and placed on ice for the t = 0 sample. The cells were collected by centrifugation, returned to prewarmed YPAD media, and allowed to recover in the dark. Experiments were also repeated where cells were maintained in PBS during the recovery phase to prevent the dilution of the adducts by DNA replication (GILLETTE et al. 2001 ), and under these conditions the removal of adducts was similar in the wild type and mutant (not shown). At the specified time point cells were collected, washed in ice-cold STE (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA), and frozen at –80°. Genomic DNA was isolated using standard techniques and quantified by agarose gel electrophoresis. Approximately 100 ng of DNA was denatured in 0.3 ml of 0.4 M NaOH, 1 mM EDTA for 10 min at 65° and was applied to HybondN+ (Amersham-Pharmacia) using a slot blot manifold. The membrane was blocked in 2% nonfat milk (NFM) in TBST (50 mM Tris-HCl, ph 7.4, 150 mM NaCl, 0.05% Tween 20) for 1 hr and incubated for 1 hr at 37° with anti-thymidine dimer monoclonal antibody KTM53 (Kamiya Biomedical, Seattle, WA) prepared in 0.5% NFM in TBST at a 1:300 dilution. After washing, the membrane was incubated at room temperature with a secondary antibody/horseradish peroxidase conjugate in 0.5% NFM in TBST. The secondary antibody was detected by chemiluminescence. Western blot signals were corrected for the amount of DNA bound to the membrane, which was determined by probing the membrane with radiolabeled total genomic DNA.

View larger version (40K): In this window In a new window Download PPT slide   Figure 1. DHH1 is the ortholog of p54/RCK and is required for resistance to DNA damage. (A) Viability after exposure to UV irradiation and growth on YPD plates containing 0.015% MMS were measured in YJR218 (DHH1) and YJR219 (dhh1). Plates lacking and containing MMS were grown for 2 or 3 days at 30°, respectively. (B) Sequence conservation between Dhh1p and p54/RCK. The lengths of Dhh1p and p54/RCK are 506 and 482 amino acids, respectively. (C) Complementation of the temperature-sensitive and DNA-damage-sensitive phenotypes of the dhh1 mutant by p54/RCK expression. Fivefold serial dilutions of cultures of YJR218 and YJR219 transformed with pG1 or pG1-(HA)2-p54/RCK were spotted onto SC-tryptophan plates or the same medium containing 0.01% MMS, and one was exposed to 60 J/m2 UV irradiation after plating. Plates were placed at 30° for 2 or 3 days (DNA damage exposure and 37° conditions).

View larger version (38K): In this window In a new window Download PPT slide   Figure 2. dhh1 mutant is severely delayed in emergence from G1/S checkpoint arrest. (A, top) Wild-type (YJR218) and dhh1 (YJR219) cells were synchronized in G1 with -factor, then the -factor was removed, and aliquots were taken for FACS analysis over a 6-hr time period. (A, bottom) Wild-type and dhh1 cells were exposed to the DNA-alkylating agent methyl methanesulfonate (MMS) while arrested in G1 with -factor, and cell cycle progression after removal of -factor and MMS was followed by FACS. (B) Wild-type (YJR218) and dhh1 (YJR219) cells were subjected to the same time course as described in A, but they were treated with 60 J/m2 UV irradiation rather than MMS. (C) Reaccumulation of CLN2 mRNA is severely delayed in dhh1 cells following activation of the G1/S checkpoint by UV-induced DNA damage. Aliquots were taken for quantifying G1 cyclin mRNA by Northern blotting over the same time course as in A and B. ScR1 is a loading control.

View larger version (31K): In this window In a new window Download PPT slide   Figure 3. The protracted G1 arrest of dhh1 cells is dependent upon an intact checkpoint response. (A) Viability after exposure to UV irradiation was measured in strains carrying deletions of DHH1 and MEC1. Wild-type (sml1::URA3, YJR535), dhh1 sml1 (YJR534), mec1 sml1 (YJR533), and dhh1 mec1 sml1 (YJR531) cells are shown. All strains used contain a deletion of SML1 to preserve the viability of the mec1 and rad53 strains (ZHAO et al. 1998 ), but only the relevant, distinguishing genotypes are listed here and in the text. (B) Cells were synchronized in G1 with -factor and exposed to UV irradiation and then released from -factor and followed by FACS as in Fig 2A. (C) Samples of dhh1 and dhh1 mec1 cells were collected over the same time course, after induction of DNA damage by MMS, for analysis of CLN2 mRNA expression by Northern blotting. ScR1 is a loading control.

View larger version (24K): In this window In a new window Download PPT slide   Figure 4. DHH1 is not required for DNA damage repair. DHH1, dhh1, and rad23 cells were exposed to 50 J/m2 and allowed to recover for 30–180 min. Genomic DNA was prepared, denatured, and applied to a membrane. (A) Western blot using anti-thymidine dimer monoclonal antibody KTM53. (B) Quantification of the results. Values represent percentage of signal at t = 0 and are normalized to total DNA as described in MATERIALS AND METHODS.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

DHH1 is highly conserved and dhh1 cells are sensitive to DNA damage:
A DHH1 null mutant is viable, but grows more slowly than the isogenic wild-type strain and displays temperature-sensitive growth (STRAHL-BOLSINGER and TANNER 1993 ; HATA et al. 1998 ; Supplemental Figure 1 at http://www.genetics.org/supplemental/). We found that it is also hypersensitive to various DNA-damaging agents, including UV irradiation and the DNA alkylating agent MMS (Fig 1A).

Dhh1p displays a remarkably high identity (68%) and similarity (82%) to its human ortholog p54/RCK over the central 400 amino acids of the yeast protein (Fig 1B). This unusually high degree of conservation argues that the functions of these genes have been highly conserved throughout evolution. To address this, the coding sequence of p54/RCK was amplified and inserted into a yeast expression vector (pG1), and the resulting plasmid was introduced into cells carrying a DHH1 null allele. The results shown in Fig 1C reveal that expression of p54/RCK can complement the temperature-sensitive growth and DNA-damage-sensitive phenotypes of the dhh1 cells, suggesting that the two proteins perform many of the same functions in their respective organisms. These results provide strong evidence that DHH1 is indeed the ortholog of the putative oncogene p54/RCK.

dhh1 cells are defective in G1/S DNA-damage checkpoint recovery:
Because DNA-damage sensitivity is often associated with defects in checkpoint-induced cell cycle arrest and because DHH1 is genetically linked to cell cycle control (MORIYA and ISONO 1999 ; REESE and GREEN 2001 ), we investigated the cell cycle characteristics of the dhh1 strain using FACS analysis. The FACS profile of an asynchronous population of dhh1 cells is indistinguishable from that of the wild type, indicating that they are not delayed at any particular point in a normal, uninterrupted cell cycle and that the slow-growth phenotype is due to generally slowed progression throughout the cell cycle (Supplemental Figure 1). We next investigated the checkpoint response of dhh1 cells after inducing DNA damage. Populations of wild-type and dhh1 cells were synchronized in the G1 phase of the cell cycle, using the mating pheromone -factor. Half of each population was exposed to the DNA-damaging agent MMS, and then the -factor and MMS were removed by washing with fresh media.

In the absence of DNA damage, dhh1 cells required an additional 15–20 min to emerge from -factor-induced arrest relative to the wild-type cells (Fig 2A, top), consistent with the overall reduced growth rate of the strain. Wild-type cells displayed a delay in G1/S progression after exposure to DNA damage due to activation of the G1/S checkpoint, but these cells resumed cell cycle progression by 90–120 min after release, and approximately half the cells entered G2 by 180 min (Fig 2A, bottom left). Like the wild type, the dhh1 mutant activated its checkpoint and arrested, indicating no defects in checkpoint activation. However, the dhh1 cells showed a severely protracted G1 arrest and a somewhat slowed S-phase compared to that of the wild-type strain (Fig 2A, bottom right). dhh1 cells began to resume progression into S-phase only at 4 hr after release and required 6 hr for a small population of cells to enter G2. Similar results were observed in UV-treated cells (Fig 2B). This phenotype is specific for DNA-damage-induced checkpoints because dhh1 cells did not show an extensive delay relative to wild-type cells in emerging from G1/S arrest in the absence of DNA damage (Fig 2A, top right). Thus, dhh1 cells are competent for cell cycle arrest, but appear to be unable to subsequently recover from the arrest. This behavior is in contrast to that of known DNA-damage cell cycle checkpoint mutants, which fail to arrest after DNA damage (HARTWELL and KASTAN 1994 ; WEINERT et al. 1994 ; LYDALL and WEINERT 1995 ; ZHOU and ELLEDGE 2000 ).

The FACS analysis presented above indicates that dhh1 cells delayed DNA replication under conditions of DNA damage. However, this assay cannot distinguish cells that failed to progress through the G1/S boundary from those that passed through the checkpoint and arrested prior to DNA replication. To further characterize the precise position of the protracted cell cycle arrest in dhh1 cells following DNA damage, we followed the accumulation of the mRNA for the cyclin CLN2 over the same arrest-and-release time course. A strong but transient burst of expression of the G1 cyclins CLN1 and CLN2 is part of the cascade of gene expression that defines passage through the G1/S transition (START; LEVINE et al. 1995 ; NASMYTH 1996 ). It has been shown that the activation of the G1/S checkpoint affects its cell cycle delay at least in part by causing a delay in the reaccumulation of CLN2 mRNA (SIDOROVA and BREEDEN 1997 ) and, therefore, measuring CLN2 expression is a direct way of monitoring release from the G1/S DNA-damage checkpoint. Thus, if the failure in cell cycle progression observed for dhh1 cells is truly a failure in reemergence from checkpoint arrest, then it should be characterized by a protracted delay in CLN2 reaccumulation. This is what was observed upon measuring CLN2 reaccumulation by Northern blotting. In the absence of DNA damage, the peak of CLN2 expression is delayed 20–30 min in the dhh1 strain compared to the wild type, as might be expected from the slight delay observed in the FACS profiles shown in Fig 2A (Fig 2C). However, following UV treatment, the peak of CLN2 mRNA is delayed 120–180 min in the mutant cells compared to the wild-type cells, again consistent with the FACS data. These data indicate that the dhh1 cells are delayed at the G1/S boundary prior to START, at the cell cycle position of the G1/S checkpoint arrest.

The protracted G1/S arrest of dhh1 cells is checkpoint dependent and not due to repair defects:
If indeed the protracted arrest of the dhh1 cells is due specifically to an inability to recover from a checkpoint arrest, then one prediction is that an intact checkpoint response should be required in order to observe the protracted cell cycle delay phenotype. To test this prediction, we began by isolating a double mutant in which MEC1 was deleted in a dhh1 background. Viability of the dhh1 mec1 strain was preserved by also deleting SML1 (Suppression of Mec1 Lethality), an inhibitor of ribonucleotide reductase, the deletion of which preserves viability of deletion mutants for the essential checkpoint genes MEC1 and RAD53 (ZHAO et al. 1998 ). The double mutant showed no significant synthetic effect with regard to DNA-damage sensitivity, with viability after exposure to UV irradiation approximately the same as the viability of the mec1 strain (Fig 3A). This places DHH1 in an epistasis group with MEC1 with regard to the checkpoint response and supports the notion that the role of DHH1 in the checkpoint response may be in recovery from the checkpoint arrest. To further characterize the checkpoint behavior of the dhh1 mec1 strain, the arrest and release time course described in Fig 2 was repeated with the double mutant, using UV irradiation as the DNA-damaging agent. The protracted delay observed in cells lacking DHH1 is indeed dependent upon an intact checkpoint, as might be expected if the role of DHH1 is specific to checkpoint recovery. The dhh1 mec1 strain was greatly accelerated through the checkpoint, as measured by FACS analysis, compared to the dhh1 strain. The fact that deletion of MEC1 failed to completely reverse the dhh1-induced delay (Fig 3B, compare bottom right to top right) is likely due to residual checkpoint activity in MEC1 deletion mutants (MORROW et al. 1995 ; SANCHEZ et al. 1996 ). Progression through START was also measured by CLN2 mRNA accumulation in the double mutant, as described in Fig 2 (Fig 3C). Again, the Northern results support the suggestion by FACS that inactivation of the checkpoint response suppresses the requirement for DHH1 in passage through START following DNA damage. These results further support the model that DHH1 plays a specific role in checkpoint recovery, following checkpoint activation and cell cycle arrest.

One possibility suggested by the results described above is that DHH1 plays a role in repairing DNA damage, such that deletion of DHH1 impairs repair, resulting in persistence of the damage-signaling cascade that mediates G1/S checkpoint arrest. To investigate this possibility, we followed repair of UV photoproducts in wild-type and dhh1 cells, using an antibody against thymidine dimers. As a control, repair was also followed in rad23 cells, which are known to be defective for the nucleotide excision repair pathway (GILLETTE et al. 2001 ; Fig 4). The dhh1 strain shows no defect compared to the wild type in the repair of UV-induced damage, while the control rad23 strain is obviously defective in repair as measured by this assay. These results suggest that the role of DHH1 in checkpoint recovery is at the level of regulation of cell cycle progression, rather than at the level of actually repairing DNA damage.

The checkpoint recovery defect of dhh1 cells is specific to the G1/S checkpoint:
Many DNA-damage checkpoint genes characterized to date in yeast regulate the G1/S, S, and G2/M DNA-damage checkpoints (HARTWELL and KASTAN 1994 ; WEINERT et al. 1994 ; LYDALL and WEINERT 1995 ; ZHOU and ELLEDGE 2000 ). To determine whether the severe delay in emergence from G1/S checkpoint arrest observed for the dhh1 strain is specific to the regulation of START or a general defect in checkpoint recovery, we examined the recovery of dhh1 cells from the replication (S) and G2/M checkpoints.

To examine the activation of and release from the S-phase replication checkpoint, cells were arrested in S-phase with hydroxyurea (HU), and cell cycle progression was followed by FACS analysis after its removal. dhh1 cells were capable of arresting in response to HU treatment similarly to wild-type cells and upon release reentered the cell cycle at nearly the same rate as wild-type cells (Fig 5A). The mutant required 15–20 additional minutes to enter G2/M compared to the wild-type strain, but clearly the delay was not nearly as severe as what was observed at the G1/S DNA-damage checkpoint. Instead, the extended period of time required by the dhh1 cells to enter G2/M after release from HU block was similar to that observed for its emergence from -factor arrest in the absence of DNA damage. This slight delay is attributable to the overall slowed growth of this strain and likely does not indicate defects in emerging from this checkpoint specifically (see also below).

View larger version (26K): In this window In a new window Download PPT slide   Figure 5. dhh1 cells are not deficient in recovery from S phase or G2/M checkpoints. (A) The S-phase checkpoint was activated in wild-type and dhh1 cells using the replication inhibitor hydroxyurea (HU) and cell cycle progression after removal of HU was followed by FACS. (B) Wild-type and dhh1 cells were synchronized in the G2 phase of the cell cycle using the microtubule polymerization inhibitor nocodazole and exposed to UV irradiation to activate the G2/M checkpoint. Cell cycle progression following removal of nocodazole was assessed by observing bud morphology. The symbols are as follows: squares, DHH1, –UV irradiation; circles, dhh1, –UV irradiation; triangles, DHH1, +UV irradiation; diamonds, dhh1, +UV irradiation. (C) As in B except the percentage of binucleate cells was counted as a measure of progression through the G2/M checkpoint at 15-min intervals following release.

To examine the activation of and release from the G2/M checkpoint, cells were synchronized in G2/M with nocodazole, exposed to UV irradiation, and released into the cell cycle by removal of the drug. Progression out of the G2 checkpoint arrest and through M phase was monitored by calculating budding indices on the basis of counting large-budded cells (Fig 5B) and also by calculating the percentage of cells that were binucleate as visualized following 4',6-diamidino-2-phenylindole staining (Fig 5C). As previously observed for -factor and HU arrests, the dhh1 cells required an additional 20–30 min to emerge from nocodazole block in the absence of DNA damage compared to wild-type cells. However, and more importantly, they emerged from the G2/M DNA-damage checkpoint at a rate indistinguishable from that of the wild type. Thus, DHH1 is specifically required for the recovery from the G1/S DNA-damage checkpoint. Furthermore, these data provide additional support to the model that the role played by Dhh1p at the G1/S checkpoint impinges on the cell cycle regulatory machinery and not directly on damage repair. If the protracted G1/S arrest seen in dhh1 cells was due solely to defects in actually repairing DNA damage, these defects in damage repair would be likely to cause a protracted checkpoint arrest at the S-phase and G2/M checkpoints as well.

dhh1 cells are hypersensitive to additional cell cycle perturbations:
Since the role of DHH1 in recovery from G1 cell cycle arrest following DNA damage appeared to be at the level of cell cycle regulation, we wondered whether it would be possible to fully suppress the requirement for DHH1 in cell cycle reentry simply by further reducing checkpoint activity or by increasing expression of positive cell cycle progression factors such as G1 cyclins. Since the deletion of MEC1 had caused a partial suppression of the G1 delay seen in the dhh1 strain following DNA damage, we started by attempting to fully inactivate the G1 checkpoint to see if this could lead to a full suppression of the delay. Starting with a strain background lacking SML1 to preserve viability of RAD53 and MEC1 mutants (ZHAO et al. 1998 ), we attempted to isolate a triple mutant lacking DHH1, MEC1, and its partially redundant homolog TEL1 or a double mutant for DHH1 and RAD53, the kinase of which is believed to act downstream of MEC1 and TEL1 in the DNA-damage signaling pathway at G1/S (SUN et al. 1996 ). These attempts were unsuccessful unless DHH1 was supplied on a URA3 plasmid during strain construction (data not shown). After successfully isolating the dhh1 mec1 tel1 and dhh1 rad53 mutants carrying the wild-type DHH1 on a URA3 plasmid, cultures of these strains were spotted on plates containing 5-fluoroorotic acid (5-FOA) to test for viability of the mutants after the loss of DHH1 (Fig 6A). Surprisingly, fully disabling the known G1/S checkpoint activation pathway proved to be synthetically lethal with deletion of DHH1, even in the absence of exogenous DNA damage. MEC1 and RAD53 do play additional roles in cell cycle regulation besides activation of the G1/S checkpoint response, as evidenced by the fact that they are essential for cell viability regardless of damage, if the negative regulator of dNTP pools, SML1, is functional (ZHAO et al. 1998 ). It has been suggested that the essential roles of these proteins are related to dealing with minor "damage" that occurs as part of each cell cycle in the G1, S, and G2 phases of the cell cycle (ZHAO et al. 2001 ). Our observation of synthetic lethality of MEC1 TEL1 or RAD53 deletion with DHH1 deletion does not address the mechanism or elucidate the relevant cell cycle phase at which these proteins may interact. However, this observation does support the model that DHH1 contributes to the cell cycle regulatory machinery that allows the cell to deal with the cell cycle perturbations that are regularly encountered.

View larger version (33K): In this window In a new window Download PPT slide   Figure 6. dhh1 cells are hypersensitive to cell cycle perturbations. (A) Complete inactivation of G1/S checkpoint function is lethal in a dhh1 background. Strains analyzed in this figure are sml1 (YJR745), dhh1 sml1 (YJR747), rad53 sml1 (YJR727), dhh1 rad53 sml1 (YJR723), dhh1 mec1 sml1 (YJR725), mec1 tel1 sml1 (YJR749), and dhh1 mec1 tel1 sml1 (YJR726). The viability of YJR723 and YJR726 strains was isolated in the presence of DHH1 supplied on the URA3 plasmid pRS416-DHH1. These strains were then grown in the presence of 5-fluoroorotic acid (5-FOA) to evaluate their viabilities in the absence of DHH1 expression. Note that all strains contained the sml1 mutation, but this information is not indicated within the figure to highlight the relevant phenotypes. Overexpression of G1 cyclins in dhh1 cells is shown. (B) Constitutive expression of CLN2 does not rescue the dhh1 cell cycle delay phenotype. Cells were transformed with a plasmid carrying the CLN2 gene under control of the ADH1 promoter on a low-copy plasmid that allows for moderate, constitutive expression. Cells were subjected to a similar synchronization, damage, and release time course as described in Fig 2, except that UV irradiation was used as a mutagen. A small fraction of cells escape -factor block due to the constitutive expression of CLN2, but this does not obscure the analysis. (C) Overexpression of CLN3 or a derivative lacking its destruction box (CLN3DB) from the inducible GAL1 promoter contained on a high-copy-number vector is lethal in a dhh1 background, but not in a wild-type background.

We next decided to attempt to alter expression of cell cycle regulatory factors known to act specifically at the G1/S transition, to suppress the requirement for DHH1 following DNA damage. Since at least one target of the checkpoint machinery leading to cell cycle delay at G1 is CLN2 transcription (SIDOROVA and BREEDEN 1997 ), we wondered whether the checkpoint recovery defect of dhh1 cells could be suppressed by expressing G1 cyclins from an exogenous promoter. CLN2 was placed under the control of the ADH1 promoter contained on a low-copy-number plasmid, which gives constitutive, moderate levels of expression. Expressing CLN2 from the ADH1 promoter failed to accelerate the progression of the dhh1 strain through the DNA-damage checkpoint (Fig 6B). CLN2 mRNA accumulated in the dhh1 cells to levels equal to that of the wild-type strain, indicating that the failure of exogenous CLN2 expression to accelerate checkpoint progression was not due to trivial expression defects in this mutant (Supplemental Figure 2 at http://www.genetics.org/supplemental/). These results indicate that DHH1 plays a role in regulating G1/S progression that is broader than triggering G1 cyclin transcription. This notion is supported by some of the phenotypes observed in dhh1 mutants. For example, deletion of DHH1 causes phenotypes consistent with cell wall defects (HATA et al. 1998 ), and genes required for cell wall formation are, along with the G1 cyclins, activated at START (LEVINE et al. 1995 ; NASMYTH 1996 ).

Another part of the cascade of gene expression activated at START, which is upstream of CLN2 expression, involves upregulation of CLN3. CLN3 is a G1 cyclin that is expressed at low levels throughout the cell cycle, and its post-transcriptional upregulation during G1 drives the expression of CLN2 and many other genes required for progression through G1/S. Overexpression of CLN3 precociously drives cells through start, circumventing normal cell cycle control and checkpoint mechanisms (LEVINE et al. 1995 ; NASMYTH 1996 ). Furthermore, the post-transcriptional regulation of CLN3 mRNA under conditions of cell stress influences cell cycle progression (GALLEGO et al. 1997 ; POLYMENIS and SCHMIDT 1997 ; PHILPOTT et al. 1998 ). Therefore, we wondered whether CLN3 might be a target of regulation by DHH1 and whether overexpressing CLN3 could suppress the cell cycle delay phenotype seen in dhh1 cells. However, inducing overexpression of CLN3 from the GAL1 promoter severely reduced the growth and viability of dhh1 cells, and expression of a version lacking its destruction box (DB) proved lethal in this background (Fig 6C). Together with the observed synthetic lethalities in the dhh1 mec1 tel1 strain and the dhh1 rad53 strain, this result supports the model that DHH1 contributes to the balance of positive and negative signals that modulate cell cycle progression following DNA damage. While the inherent plasticity of the cell cycle may allow the cell to tolerate a strong perturbation or the loss of some part of its regulatory machinery, combinations of these insults lead eventually to cell death. The fact that loss of DHH1 shows synthetic lethality with strong cell cycle perturbations in the form of DNA damage and with loss of regulation via checkpoint deletion or cyclin overexpression implicates it as part of the regulatory network that maximizes viability by contributing to control of cell cycle progression.

dcp1 mutants are also sensitive to DNA damage:
Previous studies have suggested associations between Dhh1p and several proteins with roles in mRNA degradation in yeast, including the decapping enzyme Dcp1p (COLLER et al. 2001 ; FISCHER and WEIS 2002 ). We wondered whether the DNA-damage phenotypes observed in the dhh1 strain would also be associated with disruption of the decapping machinery.

DCP1 encodes the major yeast mRNA-decapping enzyme, and DHH1 is reported to stimulate its decapping activity (FISCHER and WEIS 2002 ). We next assayed the DNA-damage sensitivity of a dcp1 mutant and found it to be nearly as sensitive to UV irradiation and MMS as the dhh1 strain (Fig 7). Unfortunately we were unsuccessful in performing the block, damage, and release studies described in Fig 2 to assess the integrity of the G1/S checkpoint in this mutant because it arrests poorly in response to -factor (not shown). The dcp1 mutant also showed more severe growth defects than the dhh1 mutant and is inviable in some genetic backgrounds. This is consistent with DHH1 playing a stimulatory and/or regulatory role in decapping, whereas DCP1 plays an essential role. Nonetheless, these data support the notion that the DNA-damage sensitivity phenotypes observed in dhh1 cells are closely linked to the function of Dhh1p in the decapping complex.

View larger version (21K): In this window In a new window Download PPT slide   Figure 7. DNA damage sensitivity phenotype of the dhh1 strain is shared by dcp1 cells. UV and MMS sensitivity were assayed as described in Fig 1 for dcp1 (YRP1071) and its isogenic wild-type strain (YRP840). dhh1 and its isogenic wild-type strain were included in the MMS sensitivity assay for direct comparison. The dcp1 strain is nearly as sensitive as the dhh1 strain.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

DNA-damage sensitivity phenotypes are linked to mRNA metabolism:
That Dhh1p is known to colocalize with and stimulate the decapping machinery in cytoplasmic foci (FISCHER and WEIS 2002 ; SHETH and PARKER 2003 ) and also appears to interact with the mRNA deadenylation complex (HATA et al. 1998 ; COLLER et al. 2001 ; TUCKER et al. 2002 ) strongly suggests that the role of Dhh1p in G1/S checkpoint recovery is at the level of regulation of mRNA metabolism. Recently, deletion of DHH1 has also been shown to be synthetically lethal with mutations in DBP5 and DED1, DEAD-box helicases with roles in mRNA export and translation initiation, respectively (TSENG-ROGENSKI et al. 2003 ). This result also supports a model in which the major role of DHH1 is post-transcriptional. Further, that deletions of other components of the decapping and decay machinery also cause DNA-damage sensitivity phenotypes similar to that observed in the dhh1 strain suggests that the role of Dhh1p in checkpoint recovery is closely linked to its function in mRNA decapping. We have shown here that deletion of the decapping protein DCP1 causes increased sensitivity to UV irradiation. In addition, a lsm1 strain was shown to be moderately hypersensitive to UV irradiation in a genome-wide screen for deletions conferring DNA-damage hypersensitivity (BIRRELL et al. 2001 ), and pat1 cells have also been shown to be mildly hypersensitive to UV irradiation (WANG et al. 1999 ).

While our results cannot rule out a role for Dhh1p in regulating transcription in response to DNA damage, it seems unlikely that this is the case. Although physical and genetic interactions exist between DHH1 and components of the Ccr4-Not complex that has been implicated in transcriptional regulation, it seems that Dhh1p function is more closely linked to the Ccr4 deadenylase and the Dcp1 decapping complexes. Ccr4p, Pop2p, and Dhh1p all associate with the N-terminal domain of Not1p, the only essential component of the Ccr4-Not complex, while the other Not proteins, mutations of which cause the strongest transcription phenotypes, all associate with the C-terminal domain of Not1p (BAI et al. 1999 ; DELUEN et al. 2002 ; MAILLET and COLLART 2002 ). It has been proposed that the apparent functions of the Ccr4-Not complex in both transcriptional regulation and deadenylation suggest a high degree of coordinated regulation of mRNA metabolism throughout the lifetime of an mRNA, from its transcription through its destruction (MAILLET and COLLART 2002 ; TUCKER et al. 2002 ). If Dhh1p is a modulator of mRNA metabolism, it is reasonable to expect that it would associate with a complex or complexes that affect an mRNA throughout its lifetime.

There are several possible roles that a DEAD-box RNA helicase might be imagined to play in association with the deadenylation and decapping machinery, which could affect regulation of mRNA stability, regulation of the translational state of the mRNA, or both. DEAD-box helicases involved in other processes such as splicing and translation are known to be required for taking apart protein-RNA complexes so that they can be remodeled to allow for the next step in these processes (SCHWER 2001 ; TANNER and LINDER 2001 ). It might be that Dhh1p has a similar role in helping to remodel the interactions between mRNAs and their associated mRNP proteins as the status of the mRNP changes over the course of its lifetime. Indeed, several lines of recent evidence suggest that the DDX6-like DEAD-box helicases, which include DHH1, associate with mRNAs throughout the lifetime of the mRNA. In addition to the associations between Dhh1p and transcription, deadenylation, and decapping complexes in yeast, the Xenopus DHH1 ortholog Xp54 has been shown to interact with nascent transcripts in the nuclei of transcriptionally active oocytes, but to localize to the cytoplasm in transcriptionally quiescent oocytes. Furthermore, its shuttling between the nucleus and cytoplasm is developmentally regulated (SMILLIE and SOMMERVILLE 2002 ).

An attractive model is that Dhh1p and its orthologs associate with a subset of mRNAs and regulate their stability and/or translation. Such a model would suggest that efficient recovery from G1/S checkpoint arrest requires Dhh1p either to stimulate the decay or to alter the translational status of a subset of mRNAs or possibly even to perform both functions. However, the specific mRNAs that may be affected and the ways in which they are affected have yet to be determined. Studies in Drosophila (NAKAMURA et al. 2001 ) and clam (MINSHALL et al. 2001 ) have shown that the DHH1 orthologs in these organisms act to repress translation of maternal mRNAs to which they bind during early development. In Drosophila, two mRNAs, osk and BicD, that are normally silenced until they are transported into the oocyte, are prematurely translated in nurse cells when the Dhh1p ortholog is inactivated (NAKAMURA et al. 2001 ). In other organisms, no specific mRNA targets of Dhh1p have been identified. While the very specific G1/S checkpoint recovery defect associated with deletion of DHH1 may seem to be suggestive of specific mRNA targets of Dhh1p, it is also possible that G1/S-specific transcripts are simply the most sensitive among a very large number of mRNA targets of Dhh1p to this type of regulation of mRNA metabolism.

Post-transcriptional control at the G1/S boundary:
Recent evidence strongly suggests that post-transcriptional or translational control mechanisms play an important role in regulating cell cycle progression through G1/S in higher eukaryotes. Both inhibitors of G1/S progression, such as p53 and the cyclin-dependent kinase inhibitor p21 (PETER 1997 ; WANG et al. 2000 ) and proto-oncogene stimulators of cell cycle progression (LANDERS et al. 1997 ) have been shown to be regulated at the level of mRNA stability and translation. Many of these G1/S regulatory messages have naturally short half-lives and thus are particularly sensitive to regulation of mRNA stability (CHEN and SHYU 1995 ). In yeast, G1/S progression is known to be sensitive to regulation at the level of mRNA stability and translational efficiency. Mutation of the cap-binding protein, eIF4E, which destabilizes some mRNAs, leads to cell cycle arrest in G1. Interestingly, this arrest can be overcome by overexpression of CLN3 (DANAIE et al. 1999 ). The exceptionally high sequence conservation with DHH1 displayed by the human protein p54/RCK, and its ability to substitute for DHH1, indicates that a putative regulatory mechanism in which it plays a role, affected by modulation of mRNA stability, is highly conserved among all eukaryotes.

Checkpoint function and neoplastic transformation:
Appropriate response to DNA damage involves balancing checkpoint signaling leading to cell cycle arrest with mitogenic signaling leading to cell cycle reentry such that genomic damage is minimized and viability is maximized. It has long been clear that inactivating checkpoints, disrupting the balance toward mitogenic signaling, is catastrophic to cells (HARTWELL et al. 1994 ; WEINERT 1997 ). However, it is becoming clear that mitogenic signaling to allow cell cycle reentry is also essential (SHAULIAN et al. 2000 ). In higher eukaryotes, disruption of this balance can lead either to unregulated growth and neoplastic transformation or to apoptosis. Others have recently reported that deletion of DHH1 suppresses the deleterious effects of heterologously expressing the human tumor suppressor gene BRCA1 and suggest that this is due to a normal role for Dhh1p at the G1/S transition, where BRCA1 may serve in a checkpoint role in human cells (WESTMORELAND et al. 2003 ). Our findings indicate that DHH1 plays an important role in the cell cycle reentry process at the G1/S DNA-damage checkpoint, potentially helping to strike the balance between terminal arrest and inappropriate cell cycle progression. Loss of DHH1 function renders cells hypersensitive to DNA damage, apparently due to an inability of dhh1 cells to recover from checkpoint arrest. However, it also renders cells incapable of dealing with perturbations that accelerate cell cycle progression, such as full inactivation of the G1/S checkpoint or overexpression of CLN3. Interestingly, deletion of DHH1 or other genes involved in decapping in yeast has been reported to cause a range of apoptotic phenotypes (MAZZONI et al. 2003 ). These results provide new insight into the potential cell cycle regulatory mechanisms acting at the level of mRNA stability and translatability in yeast. Furthermore, they suggest that these regulatory mechanisms are highly conserved and that their disruption in human cells, via overexpression of the DHH1 ortholog, can contribute to neoplastic transformation.

	FOOTNOTES

¹ Present address: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94107.

	ACKNOWLEDGMENTS

The authors thank Dr. Roy Parker for advice and providing information on DHH1 prior to publication. We also are grateful to Todd Cohen, Drs. Parker, Steve Elledge, Rodney Rothstein, and Linda Breeden for strains used in early stages of this work and colleagues for advice and discussion regarding this article. Financial support for this project was provided by the National Institutes of Health (GM58672), the National Leukemia Research Association, a Penn State University Innovation Grant to J.C.R., and an American Society of Microbiologists Undergraduate Research Fellowship to M.B.

Manuscript received October 31, 2003; Accepted for publication January 9, 2004.

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