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Originally published In Press as doi:10.1074/jbc.M405080200 on July 22, 2004

J. Biol. Chem., Vol. 279, Issue 38, 39636-39644, September 17, 2004
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Rad53 Kinase Activation-independent Replication Checkpoint Function of the N-terminal Forkhead-associated (FHA1) Domain*

Brietta L. Pike{ddagger}§, Nora Tenis{ddagger}, and Jörg Heierhorst{ddagger}§

From the {ddagger}St. Vincent's Institute of Medical Research and the §Department of Medicine, The University of Melbourne, 9 Princes Street, Fitzroy, Victoria 3065, Australia

Received for publication, May 7, 2004 , and in revised form, July 7, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Saccharomyces cerevisiae Rad53 has crucial functions in many aspects of the cellular response to DNA damage and replication blocks. To coordinate these diverse roles, Rad53 has two forkhead-associated (FHA) phosphothreonine-binding domains in addition to a kinase domain. Here, we show that the conserved N-terminal FHA1 domain is essential for the function of Rad53 to prevent the firing of late replication origins in response to replication blocks. However, the FHA1 domain is not required for Rad53 activation during S phase, and as a consequence of defective downstream signaling, Rad53 containing an inactive FHA1 domain is hyperphosphorylated in response to replication blocks. The FHA1 mutation dramatically hypersensitizes strains with defects in the cell cycle-wide checkpoint pathways (rad9{Delta} and rad17{Delta}) to DNA damage, but it is largely epistatic with defects in the replication checkpoint (mrc1{Delta}). Altogether, our data indicate that the FHA1 domain links activated Rad53 to downstream effectors in the replication checkpoint. The results reveal an important mechanistic difference to the homologous Schizosaccharomyces pombe FHA domain that is required for Mrc1-dependent activation of the corresponding Cds1 kinase. Surprisingly, despite the severely impaired replication checkpoint and also G2/M checkpoint functions, the FHA1 mutation by itself leads to only moderate viability defects in response to DNA damage, highlighting the importance of functionally redundant pathways.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA damage, from endogenous or environmental sources, can be disastrous for cell survival and proliferation if not accurately repaired. Checkpoint pathways are vital to protect the genome as they monitor cell cycle progression and function to arrest the cell cycle in the presence of DNA damage and to activate repair. These checkpoint pathways are complex intricate signaling networks that are highly conserved among eukaryotes (reviewed in Ref. 1). Modular FHA1 phosphothreonine-binding domains are found in a wide variety of proteins and are particularly common among cell cycle and checkpoint proteins (reviewed in Refs. 2 and 3). The importance of the FHA domains in many of these proteins is becoming increasingly evident. A clinically relevant example of the importance of FHA domain function is seen in the Chk2 human tumor suppressor kinase, in which single residue substitutions in the FHA domain that interfere with Chk2 activation and signaling have been linked to cases of the Li-Fraumeni multicancer syndrome (4, 5). The FHA domain of Chk2 is required for heterodimerization and autophosphorylation in response to ionizing radiation and for the binding of Chk2 to two important DNA repair proteins, BRCA1 and Mus81 (6–9).

Rad53, the Saccharomyces cerevisiae homolog of Chk2, is essential for the checkpoint response to DNA damage and replication block signals as well as normal cell growth (reviewed in Ref. 10). Rad53 kinase activation is required for arrest at all stages of the cell cycle (G1/S, intra-S and G2/M checkpoints) depending on where the DNA damage occurs and for a replication checkpoint that monitors the progress of DNA replication (11, 12). Rad53 is also important for the relocalization, transcriptional up-regulation, and activation of proteins required for repair (13–15). Cell cycle-wide DNA damage is sensed by two overlapping pathways, one involving the Rad17-Ddc1-Mec3 complex and the other involving Rad9 (16, 17). The replication checkpoint is independent of the Rad17 and Rad9 branches, and its components (e.g. Pol2, Sgs1, Tof1, Mrc1, and Rfc5) are parts of or closely associated with the replication machinery. This facilitates a rapid response to DNA replication blocks that includes stabilization of stalled replication forks, inhibition of late origin firing, and prevention of S/M spindle elongation (reviewed in Ref. 10). In response to either of these pathways, Rad53 is activated through phosphorylation in a Mec1-dependent manner and then integrates these signals to activate effectors. Surprisingly, rad53{Delta} cells have much lower gross chromosomal rearrangement rates (27-fold increased as compared with wild type) than cells lacking Mec1 (194-fold) or another Chk2-like kinase, Dun1 (211-fold) (18). Consistent with this, it is now clear that there are additional Mec1-dependent pathways that act in parallel with Rad53, for example, involving the structurally diverse Chk1 kinase (19) or direct regulation of Pds1 (20).

In contrast to other Chk2-like kinases, Rad53 contains two FHA domains, the conserved N-terminal FHA1 and an additional FHA2 domain C-terminal to the kinase domain (21). The FHA2 is required for the interaction of Rad53 with phosphorylated upstream Rad9 in response to DNA damage and subsequent Rad53 activation (22–24). Interestingly, the FHA1 can also bind to phosphorylated Rad9 in vitro (25); however, it is still unclear whether Rad9 is a physiological target of the FHA1 and whether the roles of the two FHA domains overlap in this respect. Other suggested binding partners for the FHA1 domain include the protein phosphatase Ptc2 (26), the Cdc7 kinase regulatory subunit Dbf4 (27), the chromatin assembly factor Asf1 (28), and the recently identified checkpoint target Mdt1 (29). This indicates that multiple binding partners for the FHA domains contribute to the complex functions of Rad53. We have recently shown that the FHA domains are crucial for Rad53 DNA damage checkpoint functions as a rad53-R70AR605A allele, containing disrupted phosphothreonine-binding sites in both FHA domains, displays severe hypersensitivity and greatly reduced Rad53 activation to DNA damage and replication blocks, similar to a rad53{Delta} strain phenotype (30). In terms of requirements throughout the cell cycle, both FHA domains have specific roles during G2/M as inactivation of either FHA1 or FHA2 abolishes Rad53 activation and cell cycle arrest by the G2/M checkpoint, whereas the two FHA domains are largely redundant for Rad53 activation in asynchronous cells (30). Here, we demonstrate a novel function of the FHA1 domain by showing that it is specifically required to link activated Rad53 to downstream effectors for its established function to prevent late origin firing as part of the replication checkpoint.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Strains, Plasmids, and Cultures—To focus on the checkpoint function of Rad53, all mutant strains in this study were in the W303-1a background (with wild type RAD5) containing deletion of the SML1 gene to suppress viability defects of rad53 mutants (31). Construction of the rad53-R70A allele has been described (32). All other mutant strains were generated by standard PCR-based allele replacements with auxotrophic selection or antibiotic-resistant markers (Table I). Cultures were grown at 30 °C in 1% yeast extract, 2% peptone, 2% glucose (YPD), except for {alpha}-factor-treated cultures, which were grown at 24 °C. pYES-PTC2 was a gift from Mary-Jane Gething and Leon Helfenbaum, University of Melbourne. For overexpression experiments, cells were grown in medium lacking uracil and containing 2% sucrose or induced in 2% sucrose + 2% galactose for 6 h.


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  		TABLE I
Yeast strains used in this study

Y53 is the wild type.

 
Damage Sensitivity Assays—For plate assays, 2 µl of yeast cultures (starting A600 = 0.5 and serial 10-fold dilutions) were spotted on YPD containing 25 mM hydroxyurea (HU) or 0.02% methyl methane sulfonate (MMS) or UV-irradiated with 100 J/m2 using a Uvitec CL-508 cross-linker with a single light bulb and then plated on YPD and incubated for 3 days. For liquid assays, overnight cultures were diluted to A600 = 0.2 and grown for 3 h before the addition of MMS or HU. Aliquots removed immediately before and 3 h after MMS addition or every 2 h over a 12-h period after the addition of 100 mM HU were plated on YPD. After 3 days, survival rates were calculated as the percentage of colonies formed in damage-treated as compared with untreated samples in ≥3 independent experiments.

Intra-S Phase Analyses—Cells were synchronized in G1 with two doses of 20 µg/ml {alpha}-factor over 3 h and >95% synchrony, as confirmed by phase contrast microscopy. Cells were released into 200 mM HU, and samples were removed at 0, 30, 60, 90, and 120 min after release. To visualize replication intermediates generated by stalled replication forks, genomic DNA was subjected to denaturing agarose gel electrophoresis as described (33). The gel was washed twice for 20 min in neutralization buffer (1.5 M sodium chloride, 0.5 M Tris, pH 8.0), and the samples were transferred to Biodyne B positively charged nylon membrane (Pall Corp.) and hybridized with ARS305 and ARS501 sequences (probes described in Ref. 33) using standard procedures.

Western Blots and Kinase Assays—Overnight cultures were diluted to A600 = 0.2, and after 3 h, they were treated with 150 mM HU for 1 h or left untreated. Western blots and kinase assays were performed as described (30). For cell cycle phase Western analysis, cells were synchronized in G1 or allowed to proceed from a G1 arrest into S phase for 30 min or synchronized in G2/M with two doses of 15 µg/ml nocodazole over 3 h. PhosphorImager autoradiographs and densitometry scans of Western blots were quantified using ImageQuant software (34).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Genetic Analyses of the Interaction between the Rad53 FHA1 Domain and the Rad9 and Rad17 DNA Damage Sensor Pathways—We have previously shown that introduction of an R70A mutation into the endogenous RAD53 gene, which disrupts the FHA1 phosphothreonine-binding site without affecting domain folding, leads to moderately increased DNA damage sensitivity (32). Extending these studies, we found that the R70A mutation completely abolishes Rad53 activation and Rad53-dependent cell cycle arrest in the G2/M checkpoint (30). This role of the FHA1 could be due to its proposed interaction with Rad9 previously suggested from in vitro studies (24, 35, 36). To test this possibility, a strain combining the rad53-R70A allele with a gene deletion of RAD9 was generated and analyzed for DNA damage sensitivity. In these assays, the wild type strain had normal growth when plated on medium containing MMS or HU or after being UV-irradiated before plating on YPD, whereas the rad53{Delta} strain had greatly reduced viability in response to MMS and HU, and to a lesser extent, UV (Fig. 1). In the presence of these low concentrations of DNA-damaging agents, the FHA1 mutant strain had no obvious growth defect, consistent with its previously observed moderate damage sensitivity phenotype (30, 32). Under these conditions, the rad9{Delta} strain was also only mildly damage-hypersensitive (Fig. 1). Interestingly, when these two alleles were combined in rad9{Delta}rad53-R70A double mutants, cells became dramatically hypersensitive to DNA damage by MMS or UV treatment (Fig. 1), indicating Rad9-independent functions of the FHA1 domain. Similar results were obtained when the rad53-R70A mutation was combined with gene deletions of RAD17 or DDC1 in which disruption of the FHA1 domain severely hypersensitized both the rad17{Delta} and the ddc1{Delta} strains to DNA damage by MMS and UV (Fig. 1). In contrast to the rad9{Delta}rad53-R70A double mutant, the rad53-R70A mutation in rad17{Delta} or ddc1{Delta} also caused a synergistic hypersensitivity to the replication blocking agent HU (Fig. 1). These synthetic genetic interactions of the rad53-R70A allele with rad9{Delta}, rad17{Delta}, and ddc1{Delta} indicate that the FHA1 domain has major functions outside the cell cycle-wide checkpoint pathways.



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  		FIG. 1.
The Rad53 FHA1 domain has Rad9 and Rad17 independent functions. Shown are serial 10-fold dilutions of yeast strains, labeled along the top on YPD (top panel), YPD + 0.02% MMS, 25 mM HU, or 100 J/m2 UV (bottom panel); samples were incubated for 3 days at 30 °C. WT, wild type.

 
As the increased sensitivity of double mutants to chronic exposure to genotoxic agents could reflect either a growth defect or increased lethality, quantitative survival analyses were performed in which cells were treated for shorter times in liquid culture before replating on normal medium to allow survivors to form colonies. Again, in response to MMS, the rad53-R70A mutation severely increased the DNA damage hypersensitivity of rad9{Delta} and rad17{Delta} strains, as well as a strain lacking DDC1 (Fig. 2A and data not shown), and the HU sensitivity of rad17{Delta} (Fig. 2B). rad53-R70A also increased the MMS sensitivity of a rad17{Delta}rad9{Delta} double mutant, further demonstrating that the FHA1 domain has major Rad9- and Rad17-independent functions for DNA damage survival.



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  		FIG. 2.
Detailed analyses of the effects of rad53-R70A on the sensitivity of checkpoint mutants to acute HU and MMS exposure. A and C, survival curves of the indicated strains treated with 0.02, 0.03, and 0.04% MMS for 3 h. B and D, survival curves of strains treated with 100 mM HU for the indicated times. Error bars show standard errors. In combination-genotypes, rad53-R70A is abbreviated as R70A for clarity. WT, wild type.

 
The FHA1 Phosphothreonine-binding Site Mutation Does Not Exacerbate Rad53 Activation Defects in rad9{Delta} and rad17{Delta} Mutants—Rad53 phosphorylation in response to DNA damage correlates with activation of the checkpoint response and can be monitored by electrophoretic mobility shifts in Western blots (37, 38). Furthermore, Rad53 phosphorylation by upstream kinases Mec1 or Tel1 activates its protein kinase catalytic domain, and increased kinase activity can be detected in autophosphorylation assays (39, 40). To explore the mechanism by which rad53-R70A FHA1 phosphothreonine-binding site mutation enhances defects in upstream activating pathways, Rad53 activation was monitored by both mobility shifts in Western blots and in kinase autophosphorylation assays. Slower mobility forms of Rad53 were observed in response to both HU and MMS, and these Rad53 mobility shifts were not diminished in the rad53-R70A mutant as compared with wild type (Fig. 3A). In response to MMS treatment, Rad53 shifts were reduced in rad9{Delta}, rad17{Delta}, and rad9{Delta}rad17{Delta} mutants; however, the FHA1-R70A substitution did not further reduce Rad53 mobility shifts in these strains (Fig. 3, A and C). Likewise, rad53-R70A did not reduce Rad53 autokinase activity in otherwise wild type or these mutant strains (Fig. 3C, top panel). In fact, the Rad53-R70A isoform actually had moderately increased autokinase activity as compared with the wild type protein after DNA damage treatment (Fig. 3C). Therefore, the increased DNA damage sensitivity of checkpoint mutant strains by the rad53-R70A mutation is unlikely to be due to a synergistic defect in Rad53 kinase activation.



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  		FIG. 3.
Role of the FHA1 phosphothreonine-binding site in Rad53 activation by Rad9, Rad17, and Mrc1 checkpoint pathways. A and B, exponentially growing cultures of the strains indicated were untreated (–) or treated with 150 mM HU (H) or 0.1% MMS (M) for 1 h. Rad53 was detected by immunoblotting with anti-FHA1 domain antibody. WT, wild type. C, autoradiograph of Rad53 in situ autophosphorylation activity (top) and Rad53 Western blot loading control (middle) of the indicated strains treated –/+ 0.1% MMS for 1 h. Quantitative data for the autoradiogram are shown in the bottom panel. Arbitrary units are multiples of the background activity of wild type Rad53 in the absence of DNA damage treatment. The right-hand panels for Rad53 and Rad53-R70A are an independent experiment –/+ MMS. D, Rad53 Western analysis of wild type and Rad53-R70A isoforms in asynchronous (asynch) cells or synchronized in the G1, S, or G2 cell cycle stage as indicated. E, side-by-side comparison of HU-induced Rad53 mobility shifts in the indicated strains (top) and probing of the same membrane with a phospho-(S/T)Q (p-S/TQ))-specific antibody (middle). The arrow points to unshifted Rad53, the filled arrowhead points to the basally shifted band, and the open arrowhead points to the supershifted band. Densitometric quantitation of Rad53 phosphorylation by mobility shift analysis (gray bars) and phospho-(S/T)Q antibody (black bars) is shown below. Rad53 shifts indicate the fraction of shifted Rad53 (top panel, arrowheads) relative to total Rad53 (top panel, arrowheads + arrows). (S/T)Q phosphorylation values indicate the percentage as compared with the highest value. F, analysis of Rad9 phosphorylation by mobility shift (top) and using the (S/T)Q antibody (bottom) in the indicated strains under control conditions (–) or after HU or MMS treatment. rad53{Delta} was not treated with MMS. The Rad9 blot was probed with an equal mixture of Santa Cruz Biotechnology antibodies sc-6740 and sc-6742 at 1:250 dilution each. In some panels, rad53-R70A is abbreviated as R70A for clarity.

 
The FHA1 Domain Functions in the Replication Checkpoint Pathway—An alternative explanation for the DNA damage sensitivity phenotypes could be that the Rad53 FHA1 domain has important RAD9- and RAD17-independent DNA damage response functions in the replication checkpoint. DNA is most susceptible to damage during S phase (41). The DNA replication process generates structural intermediates containing regions of single-stranded DNA that are fragile and can mimic DNA structures found after damage. As well as sensing DNA damage, the checkpoint machinery must also differentiate between damage and replication intermediates and be able to detect failure of replication fork progression during S phase (reviewed in Ref. 42). When nucleotide pools are depleted by HU or when replication forks collide with DNA lesions, a separate replication checkpoint pathway, in addition to the S phase functions of the Rad9/Rad17 pathways (43), relays DNA replication interference signals to the central transducers Mec1 and Rad53 (44, 45). Of the proteins that function to activate Rad53 specifically in the replication checkpoint pathway, Mrc1 is suggested to have a similar role to Rad9 in the cell cycle-wide damage response (46). We therefore investigated whether the rad53-R70A-associated DNA damage hypersensitivity could be attributed to defective signaling from the replication checkpoint branch by deleting MRC1 from various relevant strains. mrc1{Delta} alone did not obviously sensitize the cells to HU, MMS, or UV at low concentrations in plate assays or higher concentrations in liquid assays (Figs. 1 and 2, C and D). Interestingly, in contrast to rad9{Delta}rad53-R70A and rad17{Delta}rad53-R70A, the mrc1{Delta}rad53-R70A double mutant displayed no increase in MMS, UV, or HU sensitivity as compared with the respective single mutants (Figs. 1 and 2, C and D). This suggests that the Mrc1 and FHA1 pathways are largely overlapping, and therefore, that the FHA1 is critical for Rad53 functions in the replication checkpoint pathway.

Similar to rad17{Delta}rad53-R70A, a rad17{Delta}mrc1{Delta} double mutant was dramatically hypersensitive to HU, MMS, and UV (Figs. 1 and 2). Therefore, rad53-R70A and mrc1{Delta} have qualitatively similar synthetic effects with rad17{Delta}. Furthermore, rad53-R70A did not enhance the phenotype of rad17{Delta}mrc1{Delta}, consistent with a function of the FHA1 in the Mrc1 pathway (Figs. 1 and 2, C and D). However, the sensitivity of the rad17{Delta}mrc1{Delta} strain to HU was considerably greater than that of rad17{Delta}rad53-R70A (Fig. 2D), indicating that the FHA1 domain is only involved in some parts of the Mrc1 pathway. Conversely, introduction of the rad53-R70A allele resulted in slightly increased sensitivity to UV and high doses of MMS in the rad17{Delta}mrc1{Delta} background (Figs. 1 and 2C), indicating that the FHA1 phosphothreonine-binding site has some MRC1-independent functions, consistent with its role in the G2/M checkpoint (30). Nevertheless, the overall striking similarity between the DNA damage sensitivities of mrc1{Delta} in the presence and absence of the rad53-R70A allele demonstrates that the FHA1 functions in the Mrc1 pathway.

The Rad53 FHA1 Domain Is Required to Regulate the Firing of Late Replication Origins—Eukaryotic DNA replication origins are classified as "early" or "late" depending on the time during S phase at which they are activated to initiate a pair of replication forks. A major function of the Mrc1/Rad53 pathway is to inhibit late replication origin firing in the presence of replication blocks (33, 47). We therefore examined whether the FHA1 domain is required for this Rad53-dependent suppression of late origin firing. Replication intermediates of undigested genomic DNA can be resolved using denaturing agarose gel electrophoresis, in which small replication intermediates can enter the gel and can be visualized as a smear after hybridization with origin-specific probes. We followed the accumulation of replication intermediates from the well characterized early ARS305 and late ARS501 origins. We chose the mrc1{Delta} strain as a control because it has previously been shown that the absence of Mrc1 or Rad53 causes identical defects in the regulation of late origin firing, but mrc1{Delta} does not share the poor growth properties of rad53{Delta} (46). In the presence of HU, no replication intermediates were detected from the late ARS501 origin in wild type cells, even 120 min after release from {alpha}-factor (Fig. 4A). Similar to previous observations (46), in mrc1{Delta} cells, ARS501 replication intermediates became obvious about 60 min after {alpha}-factor release. Interestingly, ARS501 fired in the presence of HU in the rad53-R70A strain with kinetics very similar to the mrc1{Delta} strain (Fig. 4A). When the same samples were probed for the early firing origin ARS305, there was no difference in the pattern of replication intermediates among wild type, rad53-R70A, and mrc1{Delta} strains (Fig. 4B), indicating that early origins fire similarly in all strains. As the rad53-R70A strain was as defective in preventing late origin firing in the presence of HU as the mrc1{Delta} strain that is entirely defective in this function, this result indicates that the FHA1 domain is essential for the established function of Rad53 in late origin control.



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  		FIG. 4.
The Rad53 FHA1 is required to prevent late origin firing in response to genomic stress. G1 arrested cultures were released into YPD + 200 mM HU, and cells were collected at the indicated times after release to visualize replication intermediates from ARS501 (A) and ARS305 (B) using alkaline gel electrophoresis. Numbers in the center are size standards (bases). {alpha}F, {alpha}-factor; MW, molecular weight; R.I., replication intermediates; WT, wild type.

 
The Rad53-R70A Isoform Is Hyperphosphorylated in Response to HU—Because of the requirement for the FHA1 domain in the Rad53-dependent inhibition of late firing origins in response to replicative stress, we tested whether the rad53-R70A mutation affected Rad53 activation from the Mrc1 pathway. Similar to what we observed in the rad9{Delta} and rad17{Delta} mutant backgrounds, the rad53-R70A mutation did not further reduce Rad53 mobility shifts in response to MMS or HU treatment in the mrc1{Delta} strain (Fig. 3B). In contrast to rad17{Delta}rad53-R70A, Rad53 shifts were essentially abolished in rad17{Delta}mrc1{Delta} with or without rad53-R70A (Fig. 3B), indicating that although the FHA1 genetically acts in the Mrc1 pathway, it does so by a distinct molecular mechanism. To differentiate whether the FHA1 domain is required for Rad53 activation during S phase in general, we monitored Rad53 phosphorylation in the rad53-R70A strain in response to MMS during various cell cycle stages. As observed previously (30), DNA damage-dependent Rad53 shifts were abolished by introduction of the rad53-R70A mutation in G2/M cells (Fig. 3D). However, in all other cell cycle phases, the Rad53-R70A isoform was phosphorylated similar to the wild type (Fig. 3D), indicating that the FHA1-defective Rad53 can be normally activated during S phase.

Surprisingly, in response to HU-induced replication blocks, we noticed that the Rad53-R70A isoform was actually hypershifted as compared with wild type Rad53 (Fig. 3A), despite the crucial role of the FHA1 domain in the prevention of late origin firing. Interestingly, this HU-dependent hypershift was also observed in rad9{Delta}rad53-R70A, rad17{Delta}rad53-R70A, rad9-{Delta}rad17{Delta}rad53-R70A, and mrc1{Delta}rad53-R70A strains (Fig. 3, A, B, and E). We quantified the fraction of shifted as compared with total Rad53 by densitometry. These analyses revealed that there was on average an approximate 50% increase in the amount of shifted Rad53 in strains containing the rad53-R70A allele (Fig. 3E). Beyond this quantitative difference, the emergence of the supershifted Rad53 band (Fig. 3E, open arrowhead) in rad53-R70A-containing strains represents an important qualitative difference, suggesting that the FHA1 mutation leads to hyperphosphorylation of Rad53 on additional sites. This is supported by Western blots using a phospho-(S/T)Q-specific antibody that detects Rad53 phosphorylation by upstream kinases Mec1 and Tel1 (24, 30, 48). Although the total fraction of shifted Rad53 increased only by ~50% in the rad53-R70A-containing strains, HU-induced phosphorylation on (S/T)Q sites was overproportionally increased by about 4-fold under these conditions (Fig. 3E). This suggests that normal Rad53 activation in response to HU probably involves only very few (if not just a single) (S/T)Q motifs but that Mec1/Tel1 phosphorylate several additional such sites when the pathway is hyperactivated due to downstream failure. The Rad53-R70A isoform was not hypershifted in response to HU in the rad17{Delta}mrc1{Delta} strain, in which Rad53 phosphorylation is essentially abolished (Fig. 3, B and E). Altogether, these data indicate that the FHA1 mutation not only leads to increased fractional Rad53 activation (~50% increase) but also to a more extensively increased phosphorylation stoichiometry (~4-fold) that can be redundantly achieved by either the Mrc1 or the Rad9/Rad17 checkpoint pathways.

The Rad53 hyperphosphorylation in response to HU in the various strains containing the rad53-R70A mutation was analyzed in more detail in time course and HU dose-response trials. Time course analysis revealed that although wild type and FHA1-defective Rad53 forms had similar HU-induced phosphorylation kinetics (Fig. 5A), there was an ~15-min lag in Rad53-R70A dephosphorylation after release from HU-containing to normal medium (Fig. 5, B and D). The Rad53-R70A isoform also had an increased HU sensitivity and was shifted at lower HU doses than wild type Rad53 (Fig. 5C). During DNA replication, short single-stranded DNA regions are generated in front of the replication fork, which become much longer when forks stall and then lead to checkpoint activation (reviewed in Ref. 49). As aberrant late origin firing in rad53-R70A mutants should result in an increased number of stalled forks and consequently increased single-stranded DNA, the increased HU sensitivity and hyperphosphorylation of the FHA1 domain mutant could be explained by a secondary checkpoint hyperactivation.



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  		FIG. 5.
Rad53-R70A is hyperphosphorylated in response to replication blocks. Rad53 Western blot analysis in wild type (top) and rad53-R70A (bottom) cells at 0, 15, 30, 45, 60, and 75 min after 150 mM HU addition (A), at 0, 15, 30, 45, 60, and 75 min after release from 1 h in 150 mM HU into YPD (B) or in the absence (0) or presence of 1, 5, 10, 20, 50, and 100 mM HU for 1 h (C). Densitometric quantification of Rad53 dephosphorylation after release from HU (data in panel B) (D). Data represent the relative amount of shifted Rad53 as compared with unshifted Rad53, normalized to t = 0 min. WT, wild type.

 
The finding that the Mrc1 and Rad9/Rad17 pathways can function redundantly in the HU-induced hyperphosphorylation of FHA1-defective Rad53 (Fig. 3, B and E) implies that the secondary checkpoint hyperactivation may involve components of the cell cycle-wide Rad9/Rad17 pathways. The Rad9 pathway is not activated by HU in wild type cells (46), and consequently, Rad9 is not significantly phosphorylated under these conditions (Fig. 3F). However, similar to recent results of plasmid complementation assays (28), we found that FHA1 mutation also led to increased Rad9 phosphorylation in response to HU, which approached the level of MMS-induced Rad9 phosphorylation in both the rad53-R70A and the wild type cells (Fig. 3F). In response to HU, Rad9 was also aberrantly phosphorylated to a comparable level in rad53{Delta} cells (Fig. 3F). Altogether these experiments indicate that the Rad9 pathway is maximally activated in HU when Rad53 cannot perform its downstream functions as part of the Mrc1 pathway, either when its FHA1 alone is mutated or when Rad53 is missing altogether (in the latter case, it would of course be impossible to detect hyperactivation of this pathway by Rad53 hyperphosphorylation).

The rad53-R70A-dependent Rad53 Hypershift Is Not Due to Disruption of the FHA1-Ptc2 Interaction—One of the proposed Rad53 FHA1 domain ligands is Ptc2, a member of the PP2C family of serine/threonine phosphatases (50), and the FHA1-Ptc2 interaction is thought to be important for the dephosphorylation/deactivation of Rad53 in the recovery from and adaptation to DNA damage (26, 40, 51). An alternative explanation for the HU-dependent Rad53-R70A hyperphosphorylation could therefore be that disruption of the FHA1 results in reduced dephosphorylation by Ptc2. We therefore investigated whether deletion of PTC2 had an effect on Rad53 phosphorylation in response to HU. If the Rad53 hyperphosphorylation in response to HU is due to disruption of an interaction with Ptc2, cells deleted for PTC2 should show a similar Rad53 hypershift to the rad53-R70A mutant. However, deletion of PTC2 led to only a slight increase in HU-dependent hyperphosphorylation of an otherwise wild type strain but not nearly to the level of the rad53-R70A mutation (Fig. 6A). Deletion of PTC2 also further slightly increased the hypershift of Rad53 in the rad53-R70A mutant (Fig. 6A). These data demonstrate that the hyperphosphorylation of the Rad53-R70A isoform in response to HU is largely independent of its proposed interaction with Ptc2. As an alternate approach, we overexpressed PTC2. Cells overexpressing PTC2 have been shown to dephosphorylate Rad53 and resume cell cycle progression faster than wild type cells in adaptation, and PTC2 overexpression can rescue adaptation-defective mutants (26). PTC2 overexpression led to a noticeably reduced Rad53 phosphorylation in all strains, including rad53-R70A, and was particularly efficient in mrc1{Delta} and mrc1{Delta}rad53-R70A mutants (Fig. 6B). Therefore, as PTC2 when overexpressed was able to reduce the rad53-R70A-dependent hyperphosphorylation, and as ptc2{Delta} was unable to mimic the hypershift, these results, when taken together, strongly indicate that Rad53-R70A hyperphosphorylation is not simply the result of defective kinase dephosphorylation by Ptc2.



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  		FIG. 6.
The Rad53-R70A hypershift is independent of PTC2. A, log phase cultures of the indicated strains were untreated (–) or treated with 150 mM HU (+) for 1 h. WT, wild type. B, indicated strains grown overnight in –uracil medium containing sucrose were grown for 6 h in sucrose or sucrose + galactose and then in the absence (–) or presence (+) of 150 mM HU for 1 h.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Here, we have shown that the FHA1 domain is critical for a major Rad53 checkpoint function during S phase. The rad53-R70A mutant is unable to prevent late origins from firing in response to HU (Fig. 4) despite the fact that Rad53 is actually hyperactivated under these conditions (Fig. 3), indicating that the FHA1 links activated Rad53 to downstream effectors in this context. Consistent with a role in the replication checkpoint, the FHA1 mutation dramatically hypersensitizes rad9{Delta} and rad17{Delta} strains to DNA damage with no major effects on the DNA damage sensitivity of mrc1{Delta} strains (Figs. 1 and 2).

A striking finding was that FHA1 domain inactivation paradoxically led to hyperphosphorylation of the Rad53 kinase in response to replication blocks despite resulting in a major defect in the replication checkpoint. However, our findings can be easily integrated in the following model (Fig. 7). In wild type cells, replication blocks lead to activation of the Mrc1 pathway and subsequent Rad53 activation through phosphorylation by the Mec1 kinase (Fig. 7A). One of the major checkpoint functions of activated Rad53 is to suppress the firing of late replication origins when early forks stall (33) (Fig. 7A). Here, we propose that the FHA1 domain is not required for Rad53 activation by the Mrc1 pathway, and Rad53-R70A is therefore initially activated normally in response to replication blocks (Fig. 5A). Because the FHA1 is required to link activated Rad53 to downstream effectors to suppress late origin firing, FHA1 mutants accumulate a larger number of stalled replication forks than the wild type (Fig. 7B). Consistent with a direct relationship between the number of active forks and the strength of the checkpoint response in HU or MMS (52), downstream failure of the pathway enhances the damage signal detected by the upstream Mrc1 pathway and leads to subsequent Rad53 hyperphosphorylation (Fig. 7B). A higher number of stalled forks also explains why mutant Rad53 remains shifted for slightly longer times than the wild type after removal from HU (Fig. 5, B and D) and why the FHA1 mutant is apparently hyper-responsive to lower HU concentrations than the wild type (Fig. 5C). In addition, increased accumulation of single-stranded DNA at stalled forks can also lead to secondary activation of the cell cycle-wide Rad9/Rad17 pathways and subsequent Rad53 hyperphosphorylation (Fig. 7B). In support of a model in which Rad53-R70A hyperactivation can be achieved by either the replication or the cell cycle-wide pathways, we have found that HU-induced hyperphosphorylation of Rad53-R70A was only abolished when replication and cell cycle-wide checkpoint pathways were simultaneously disrupted in rad17{Delta}mrc1{Delta}rad53-R70A cells (Fig. 3, A and E). Furthermore, phosphorylation of Rad9 as a molecular marker for activation of the cell cycle-wide pathways (24) was observed in response to HU only in rad53-R70A and rad53{Delta} mutants but not the wild type (Fig. 3F), directly demonstrating activation of cell cycle-wide pathways in response to Rad53 defects that interfere with its replication checkpoint function.



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  		FIG. 7.
Summary diagram of Rad53 FHA1 replication checkpoint functions. A, wild type Rad53 is activated in response to replication checkpoint activation via the Mrc1 pathway. Rad53 prevents late origins firing, which contributes to the checkpoint response. B, in rad53-R70A mutants, late origin firing is not prevented, leading to further activation by the Mrc1 pathway and secondary activation by the cell cycle-wide pathways.

 
Our data indicate an important mechanistic difference in the role of the N-terminal FHA domains in the activation of Rad53 as compared with its Schizosaccharomyces pombe counterpart Cds1. In S. pombe, Mrc1 binding to the FHA domain is required for Cds1 activation by replication blocks (53). The following data indicate that this is not the case for S. cerevisiae Rad53. First, the time course of Rad53-R70A activation in response to HU was identical to the wild type (Fig. 5A); if Mrc1-dependent activation of Rad53 was defective, one would expect that compensatory activation by the cell cycle-wide pathways would occur with a noticeable delay similar to that observed in mrc1 mutants (46, 54). Second, Rad53-R70A was still efficiently hyperphosphorylated in rad9{Delta}rad17{Delta} mutants (Fig. 3A) that are left with only the Mrc1 pathway for Rad53 activation. Third, in contrast to the two-hybrid interaction between S. pombe Mrc1 and the Cds1 FHA domain (53), we could not detect a direct interaction between the Rad53 FHA1 domain (or FHA2) with Mrc1 in yeast two-hybrid assays (data not shown) under conditions in which the FHA1 readily interacts with Mdt1 (29).

In addition to the defective origin regulation shown here, the FHA1 domain also seems to be required for checkpoint suppression of S/M spindle elongation in the presence of replication blocks, as demonstrated recently in plasmid complementation assays using a slightly different rad53 mutant (p-rad53-R70A/N107A) (28). Despite these major checkpoint defects, rad53-R70A mutants, and also mrc1{Delta} and mrc1{Delta}rad53-R70A mutants, are not very sensitive to HU or MMS (Figs. 1 and 2). It has been proposed that failure to stabilize stalled replication forks is a major reason for cell death during S phase (41). For example, rad53-21 mutants that are unable to prevent fork collapse are considerably more HU-sensitive than mrc1 mutants (46). Likewise, mec1–100 mutants that are essentially deficient in activation of the three major checkpoint signaling pathways are much less MMS-hypersensitive than mec1{Delta} cells, presumably because this allele is able to prevent high rates of fork breakdown (41, 55). The substantial viability of rad53-R70A therefore suggests that the FHA1 domain is not required for the Rad53-dependent stabilization of stalled replication forks, even if the number of stalled forks increases due to defective late origin suppression. This indicates that the FHA1 is not required for all downstream functions of Rad53 during S phase, and as a consequence, it is not surprising that other effector pathways are even enhanced due to hyperactivation of Rad53-R70A, for example, Dun1 kinase activation and HU-induced RNR3 mRNA transcription (30).

What could be the critical Rad53 FHA1 domain target involved in inhibition of late replicating origin firing? The most plausible candidate would be the Cdc7 kinase regulatory subunit Dbf4 that can directly bind to the FHA1 domain in co-immunoprecipitation and yeast two-hybrid assays (27). Cdc7-Dbf4 is required for initiation of DNA replication (56, 57) and a likely Rad53 target to inhibit late origin firing, as activated Rad53 can phosphorylate Cdc7-Dbf4 and inhibit its kinase activity in vitro (58, 59). In vivo, Dbf4 is phosphorylated and displaced from chromatin in a Rad53-dependent manner in response to HU to prevent Cdc45 and polymerase {alpha} loading onto origins and new origin activation (60, 61). Mcm2–7 are suggested to be direct targets of the Cdc7-Dbf4 kinase from in vitro studies (58, 59, 62), and it is speculated that their phosphorylation by Cdc7-Dbf4 may allow Mcm2–7 to bind Cdc45 required for replication initiation (42). The interaction of Dbf4 with the FHA1 may therefore be required to bring the Rad53 kinase close to Cdc7-Dbf4 for its inhibitory phosphorylation in the intra-S checkpoint (27). Unfortunately, there are currently no dbf4 alleles available that separate its checkpoint functions from essential DNA replication functions. However, the two-hybrid Rad53-FHA1-Dbf4 interaction was not fully abolished by FHA1 R70A mutation, and Dbf4 could also interact with the Rad53 FHA2 domain in this system (27). Considering the importance of the FHA1 phosphothreonine-binding site for our phenotypes, the possibility of another critical Rad53 FHA1 target to inhibit late origin firing can therefore not be excluded. For example, S phase-specific phosphorylation of the B subunit of polymerase {alpha}-primase is also essential for initiation of DNA replication origins (63) and is inhibited in a Rad53-dependent manner in response to HU or MMS (39). Undoubtedly, it will be most interesting to identify the direct ligand of the FHA1 that links activated Rad53 to origin regulation in the replication checkpoint.

It is widely accepted that DNA damage checkpoint pathways delay the cell cycle to allow extra time for DNA repair to prevent potentially disastrous consequences of propagating unstable genomes to daughter cells. It is now clear that inactivation of the Rad53 FHA1 domain abolishes at least three major cell cycle checkpoint responses: control of late origin firing (Fig. 4) and S/M spindle elongation (28) as part of the replication checkpoint, as well as G2/M nuclear division (30). Despite these severe cellular checkpoint defects, the rad53-R70A allele by itself, strikingly, results only in a very moderately increased DNA damage vulnerability. However, combination of the FHA1 mutation with disruption of the Rad9/Rad17 pathways, or simultaneous FHA2 domain defects (30), leads to dramatically increased DNA damage hypersensitivity. This is reminiscent of the increase in genomic instability observed when rad53{Delta} is combined with deletion of RAD24 that is believed to act in concert with the Rad17 complex (18). Altogether, this illustrates that a multitude of separate checkpoint effector functions at staggered time points throughout the cell cycle promotes a remarkable `buffering`capacity for the loss of a limited number of individual cell cycle arrest functions as long as some checkpoints remain intact. The high degree of redundancy in yeast checkpoint pathways could be used as a lesson for relevant mutations in the corresponding human tumor suppressor genes. For example, mutations in the mammalian Rad53 ortholog Chk2 lead to only modest DNA damage response defects (64) and a relatively low cancer penetrance (65), probably because Chk2 is extensively backed up by alternative cell cycle arrest pathways (66). In contrast, BRCA1 and BRCA2 mutations lead to high penetrance breast cancer predisposition (67), most likely because they are essential for the function of a single unbuffered homologous recombination repair pathway in response to DNA double strand breaks (68).


   FOOTNOTES
 
* This work was supported by an Australian Postgraduate Award (to B. L. P.) and grants and a senior research fellowship from the National Health and Medical Research Council of Australia (to J. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel.: 61-3-9288-2480; Fax: 61-3-9416-2676; E-mail: bpike{at}svi.edu.au.

1 The abbreviations used are: FHA, forkhead-associated; YPD, 1% yeast extract, 2% peptone, 2% glucose; HU, hydroxyurea; MMS, methyl methane sulfonate. Back


   ACKNOWLEDGMENTS
 
We thank Rodney Rothstein, Mary-Jane Gething, and Leon Helfenbaum for yeast strains and plasmids and members of our laboratory for helpful discussions.



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