Disruptions of mechanisms preventing rereplication are additive.
Multiple mechanisms that prevent rereplication target the components of the pre-RC (ORC, Cdc6, and MCMs) (11
). Strains in which several of these mechanisms are disrupted have been generated previously (45
). These disruptions included removal of hypothetical Cdk phosphorylation sites in Orc2 (ORC2-ps
; S16A, T24A, T70A, T174A, S188A, and S206A) (45
) and Orc6 (ORC6-ps
; S106A, S116A, S123A, and T146A) (45
), introduction of an artificial nuclear localization sequence (NLS) in Mcm7 (MCM7-NLS
), stabilization of Cdc6 by N-terminal truncation (CDC6ΔNT
), and removal of a Clb5-binding RXL motif in Orc6 (ORC6-rxl
). The removal of these controls increased the cell's propensity to rereplicate during a nocodazole arrest (45
). With this study, we found that the same is true in the absence of nocodazole, as shown in Fig. , and we demonstrate here the additive character of the mutations on the extent of rereplication. In cells expressing stabilized Cdc6 and constitutively nuclear MCMs, the disruption of cyclin-Cdk-dependent regulation of ORC subunits induced phenotypes ranging from a significant accumulation of cells with 2C DNA (Fig. , panels 2 and 3) to extensive rereplication (Fig. , panels 5 and 6, and C). Rereplication was not detectable by FACS in the absence of MCM7-NLS
(data not shown), consistent with results obtained in the presence of nocodazole (45
). Therefore, mechanisms preventing rereplication at the level of the pre-RC are additive and not simply redundant, as rereplication appears when any one mechanism is disrupted. This lack of complete redundancy is consistent with the argument that multiple overlapping mechanisms have evolved by natural selection to control rereplication.
We noted that extensive rereplication as shown in Fig. required the integration of multiple copies of GAL-CDC6ΔNT-HA. In this paper, we refer to the multiple integration allele as GAL-CDC6ΔNT-HAm. We introduced this allele into our strains by crossing, thus maintaining the same copy number within experiments. In other contexts, we used a single integration allele (GAL-CDC6ΔNT-HAs). In the presence of mutations in ORC2, ORC6, and MCM7, GAL-CDC6ΔNT-HAs caused cells to accumulate with an approximate 2C DNA content instead of causing enough rereplication to allow its detection by FACS (data not shown). This suggests that Cdc6ΔNT-HA became limiting with only a single copy of the GAL promoter construct; we do not know how much Cdc6 is produced by this construct in comparison to endogenous levels.
Rereplicating cells show a discrete cell cycle arrest.
The induction of rereplication in cycling cells caused them to arrest with a large bud and a short spindle, as detected by Tub1-GFP fluorescence. This is shown for a strain with the genotype GAL-CDC6ΔNT-HAm MCM7-NLS ORC6-ps,rxl ORC2-ps TUB1-GFP as an example in Fig. . Consistent results were obtained by staining of the DNA with DAPI (4′,6′-diamidino-2-phenylindole) or propidium iodide, which showed mostly undivided nuclei (see Fig. S2 in the supplemental material; also data not shown). This morphological phenotype indicates that rereplicating cells do not undergo an abortive mitosis. A cell cycle arrest characterized by large-budded cells with undivided nuclei and short spindles could have been induced by a DNA damage response, the intra-S checkpoint, or the spindle attachment checkpoint. We were interested to determine if any checkpoint pathways were activated in rereplicating cells.
Systematic genetic screen for genes specifically required for the survival of cells with compromised rereplication controls.
In order to determine how cells respond to the loss of rereplication controls and to determine if they activate a specific checkpoint pathway, we conducted a systematic synthetic gene array (SGA) analysis based on a published method (62
). Because extensive rereplication as detected by FACS kills the cells, even in an otherwise wild-type background (68
), we sought to conduct our screen using a background that combines only a few mutations disrupting mechanisms preventing rereplication. Our previous study showed that the proliferation of ORC6-rxl CDC6ΔNT
strains was impaired (68
). We hypothesized that this background would increase the frequency of events leading to reinitiation. We suspected that ORC6-rxl CDC6ΔNT
strains accumulated inappropriate chromatin structures resulting from an increased tendency of pre-RCs to engage in a step towards firing. As discussed below, these structures could result from the reassembly of initiation complexes, from aberrant unwinding at origins, or from overt reinitiation. For the last case, there must be limited rereplication, since we did not observe DNA content significantly beyond 2C by FACS (see below).
An alternative interpretation of the poor viability of ORC6-rxl CDC6ΔNT
strains is that these mutations impair pre-RC formation or function, rather than promoting excessive or inappropriate pre-RC formation or function. We previously showed that ORC6-ps
cells were able to maintain plasmids almost as efficiently as ORC6-wt
cells in an assay sensitive to the efficiency of pre-RC formation (68
). Using the same assay, we found no significant plasmid maintenance defect in GAL-CDC6ΔNT-HAs
cells cultivated in galactose-containing medium (4.2% ± 0.4% [mean ± standard deviation] loss/generation) compared with wild-type cells grown under the same conditions (3.1% ± 1.5% loss/generation). We confirmed that ORC6-ps
cells have no significant defect (4.6% ± 1.1% loss/generation) in this assay. We found a slightly increased plasmid maintenance defect for ORC6-ps
cells (6.8% ± 0.8% loss/generation). In preliminary experiments, we detected at least partial rescue of this defect by adding multiple origins to the reporter plasmid (25
), consistent with the loss being due to a defect in pre-RC formation or function (data not shown). Since the only known mechanistic effect of these mutations is the loss of Cdk control over reassembly of the pre-RC (45
), aberrant pre-RC formation may cause the plasmid loss phenotype by some indirect mechanism. Alternatively, these results may reflect secondary effects of the mutations on other aspects of origin function (for example, the ORC6-ps
mutation may partially inhibit MCM loading or replication initiation). In this report, we will follow the former interpretation and assume that the effects of these mutations are due to a loss of Cdk control over reassembly of a pre-RC after initiation within a single S phase; however, the possibility of subtle defects in other aspects of origin function cannot be ruled out and is a caveat to our conclusions.
If reinitiating cells require some defined pathways for survival, these pathways would be detected by our synthetic lethal analysis. We screened deletions of nonessential genes that would be lethal in the ORC6-rxl GAL-CDC6ΔNT-HAs
backgrounds on YEP-Gal. In this medium, the starting strains show only slightly slower proliferation than the wild type. In most or all cases in which an additional synthetic lethal deletion was added, the removal of putative Orc6 Cdk phosphorylation sites (“ps
” mutations) added to the severity of the genetic interactions caused by the ORC6-rxl
mutation (see below; this is consistent with the initial characterization of the ORC6-rxl
mutation in reference 68
). We therefore used both backgrounds to increase the dynamic range of our screen, and we collectively refer to them as ORC6-
. We carried out the SGA analysis by systematically crossing strains from deletion strain collections and selecting for the progeny with the genotype ORC6-rxl GAL-CDC6ΔNT-HAs geneXΔ
(see Materials and Methods). ORC6-
cells on YEP-Gal proliferate faster than ORC6-
cells on YEPD (68
) (see below). For that reason, we could detect synthetic lethality but less efficiently detect rescue in the ORC6-
cells on YEP-Gal. All of the interactions obtained from the screen were then tested against GAL-CDC6ΔNT-HAs
, and -ps
) query strains on glucose- and galactose-containing media; this secondary screen identified mutations that interacted specifically with the ORC6
alleles, with CDC6ΔNT
, or with the two in combination. Because genes recovered in the first part of the screen tended to be involved in DNA metabolism and cell cycle regulation, we included a set of candidate genes involved in these functions based on the GO annotations in the Saccharomyces
Genome Database (http://www.yeastgenome.org/
) in the second round of testing (see Table S1 in the supplemental material for a list of all genes tested in the second screen). After the second round, the identities of all gene deletions were tested by PCR. All hits obtained in the second round of testing were then confirmed by serial dilutions of the relevant strains and their control genotype strains (obtained by tetrad dissection) on YEPD and YEP-Gal (see Fig. ; see also Fig. S1 in the supplemental material). A complete list of synthetic lethal gene deletions is provided in Table . Some of the synthetic lethal interactions were additionally confirmed by a tetrad analysis using the ORC6-rxl
(instead of GAL-CDC6ΔNT-HAs
) alleles with endogenous levels of Cdc6ΔNT expression (see Table S2 in the supplemental material).
Genetic interactions obtained from screening with ORC6-(ps)rxl GAL-CDC6ΔNT-HAs strains
We also tested the effect of introducing additional candidate deletions and mutations in checkpoint pathways into ORC6-rxl CDC6ΔNT
cells (with CDC6ΔNT
under the control of the endogenous CDC6
promoter). ORC6-rxl CDC6ΔNT
cells proliferate slowly on glucose-containing medium (68
). We were surprised to discover that deletion of the checkpoint genes MRC1
, and CSM3
actually rescued the sickness and inviability of those cells (see Fig. and Table ; see below), in contrast to the many deletions of checkpoint genes that caused synthetic lethality (Table ). The proliferation defect of the ORC6-rxl GAL-CDC6ΔNT-HAs
strain on galactose medium was subtle, preventing us from screening systematically for additional gene deletions that rescue this background.
The genetic interactions identified in our screen suggest that specific DNA damage response and repair genes are crucial in cells with compromised rereplication controls. Our screen also uncovered several strong interactions with cell cycle regulators; these interactions will be characterized elsewhere. As is typical with such screens (e.g., see reference 62
), we also found interactions with some genes thought to function in apparently completely unrelated pathways (Table ; for example, EDE1
, which functions in endocytosis). We have no explanation at present for these positive results. A few genes interacted with ORC6-rxl
alone (Table ).
Most genes/proteins uncovered in our screen were already known to interact genetically or physically in a tight cluster (Fig. ). Some of the other genes not found in this cluster are nevertheless thought to function in the same pathways or to belong to the same epistasis groups. This clustering suggests the specificity of our genetic screen and of the pathways involved in response to compromised rereplication controls (Fig. , inset).
FIG. 2. Genes found to interact with mutations compromising rereplication controls (ORC6-rxl GAL-CDC6ΔNT-HAs) are known to interact in a network. Clusters of genes that caused synthetic lethality or rescued ORC6-rxl GAL-CDC6ΔNT-HAs strains (Table (more ...) Dependence on MEC1.
Strikingly, many genes involved in the DNA damage response were required in the ORC6-
background (Table ; Fig. ). MEC1
encodes a kinase required for the DNA damage response (reviewed in reference 46
is essential, and therefore its deletion was not present in the collection of haploid deletion strains that we screened. The deletion of MEC1
is rescued if SML1
is also deleted. SML1
encodes an inhibitor of ribonucleotide reductase, and its deletion increases the nucleotide pool, eliminating the reliance on MEC1
in the absence of overt DNA damage (72
). With a tetrad analysis, we observed that ORC6-rxl CDC6ΔNT mec1 sml1
spores were inviable, while ORC6-rxl CDC6ΔNT MEC1 sml1
spores were often viable and slow-proliferating (see Table S2 in the supplemental material; also data not shown), like ORC6-rxl CDC6ΔNT MEC1 SML1
). ORC6-rxl GAL-CDC6ΔNT-HAs
cells also depended on MEC1
when plated on galactose medium (Fig. ). The deletion of TEL1
, the MEC1
homolog thought to act as a sensor of DNA damage in parallel with MEC1
), had little or no effect on the viability or proliferation rate of the ORC6-rxl CDC6ΔNT
cells (see Table S2 in the supplemental material; also data not shown).
Mec1 is required for all known DNA damage checkpoints and for the response to replication defects (46
). Mec1-dependent responses can regulate replication fork progression, inhibit late origin firing, and prevent spindle elongation, leading to a reversible cell cycle arrest (34
). DNA damage responses initiated at the G1
/S transition, during S phase, or after S phase are thought to be mediated by distinct Mec1-dependent molecular pathways (48
). To explore which of these functions was critical in the ORC6-rxl CDC6ΔNT
background, we used the mec1
allele, which is a separation-of- a function allele (48
). The mec1
mutant is defective in the G1
/S and intra-S DNA damage checkpoints but is competent at delaying nuclear division after UV irradiation in G2
/M checkpoint) and preventing damage-induced replication fork collapse (48
). In a tetrad analysis, the mec1
allele failed to kill or rescue ORC6-rxl CDC6ΔNT
spores (Table S2 in the supplemental material). ORC6-rxl CDC6ΔNT mec1
spores formed slow-growing colonies, like ORC6-rxl CDC6ΔNT
spores (data not shown). We concluded that ORC6-rxl CDC6ΔNT
cells require Mec1 to signal a G2
/M checkpoint and/or to prevent replication fork collapse but that Mec1 is not required in this context to signal a G1
/S or intra-S checkpoint.
Loss of rereplication control induces Ddc2-GFP foci.
Ddc2 associates with Mec1 in a complex that is recruited to sites of DNA damage (39
). Ddc2-GFP allows the visualization of this recruitment by the formation of nuclear foci upon induction of DNA damage (39
). We introduced a DDC2-GFP
allele into our strains to test whether Ddc2-GFP foci would be detectable following a loss of rereplication control. We found that GAL-CDC6ΔNT-HAm MCM7-NLS ORC6-ps
rereplicating cells accumulated Ddc2-GFP foci when the expression of Cdc6ΔNT-HA was induced (Fig. , strain 2). An increase in Ddc2-GFP foci could also be detected in GAL-CDC6ΔNT-HAm MCM7-NLS
cells expressing ORC6-rxl
(Fig. , strain 3) or ORC6-ps
(Fig. , strain 4), but not in equivalent cells expressing ORC6-wt
(Fig. , strain 5). Even GAL-CDC6ΔNT-HAs ORC6-rxl
cells (Fig. , strain 6) accumulated a significant level of Ddc2-GFP foci when the expression of Cdc6ΔNT-HA was induced. GAL-CDC6ΔNT-HAs ORC6-rxl
cells do not overtly rereplicate and are able to proliferate in galactose-containing medium (see above). These results are consistent with the dependence of GAL-CDC6ΔNT-HAs ORC6-rxl
cells on Mec1 for survival and further suggest that a loss of rereplication control triggers a Mec1-Ddc2-dependent DNA damage response that promotes survival.
FIG. 4. Strains with compromised rereplication controls accumulate Ddc2-GFP foci. (A) Cells were grown in YPD and transferred to YPR for 8 h, and then galactose (3%) was added. Cells were visualized after 3 h for Ddc2-GFP foci by DeltaVision microscopy. In addition (more ...) Rescue by MRC1 and TOF1.
Mrc1 is known to function both in mediating the intra-S checkpoint and in DNA replication (2
), in addition to a function in sister chromatid cohesion (71
). Consistent with the absence of a genetic interaction between ORC6-rxl CDC6ΔNT
, as discussed above, the deletion of MRC1
inflicted no synthetic lethality on ORC6-
cells (data not shown). This result supports the conclusion that these cells do not rely on the intra-S checkpoint. Instead, as noted above, the deletion of MRC1
restored the normal proliferation of ORC6-rxl CDC6ΔNT
cells in tetrad analysis (Fig. ; see Table S1 in the supplemental material). ORC6-rxl CDC6ΔNT mrc1
spores were fully viable and proliferated at a rate close to that of wild-type cells, unlike the semi-inviable ORC6-rxl CDC6ΔNT MRC1
background (see Table S2 in the supplemental material) (68
). Similar results were obtained by deleting TOF1
, which encodes a protein that associates and moves with Mrc1 at replication forks (27
), and by deleting CSM3
, which encodes a protein that interacts with Tof1 (36
) (Fig. ; see Table S2 in the supplemental material). Therefore, Mrc1, Tof1, and Csm3 are required for a function that slows down the proliferation of ORC6-rxl CDC6ΔNT
We next tested whether the checkpoint function of Mrc1 was responsible for the slow growth of ORC6-rxl CDC6ΔNT
cells. We used the separation-of-function allele mrc1-AQ
, in which all the consensus sites for phosphorylation by Mec1 (SQ and TQ) have been mutated to AQ motifs, uncoupling Mrc1 from Mec1 and disrupting the intra-S checkpoint (47
). Using tetrad analysis, we observed that ORC6-rxl CDC6ΔNT mrc1-AQ
cells proliferated slowly, like ORC6-rxl CDC6ΔNT
cells (i.e., mrc1AQ
did not rescue ORC6-rxl CDC6ΔNT
cells) (see Table S2 in the supplemental material). Therefore, the higher proliferation rate of ORC6-rxl CDC6ΔNT mrc1
cells was not due to the removal of the intra-S checkpoint signaling function of Mrc1 but was due to the removal of its replication function or sister chromatid cohesion function. Since Tof1 and Csm3 are thought to function together with Mrc1 (27
), we expect that the same conclusion will apply to all three proteins.
Dependence on DDC1, RAD17, and RAD53.
The 9-1-1 complex, so called because it is composed of Rad9, Rad1, and Hus1 in S. pombe
, is made up of Rad17, Ddc1, and Mec3 in S. cerevisiae
. This ring-shaped complex is loaded at sites of damage in a Rad24-dependent manner and acts there as a sensor (30
). The deletion of RAD17
rendered ORC6-rxl GAL-CDC6ΔNT-HAs
cells inviable in our screen and in plating experiments (Table ; see Fig. for RAD17
and Fig. S1 in the supplemental material for DDC1
). This result was also confirmed with ORC6-rxl CDC6ΔNT
spores in a tetrad analysis assay (see Table S2 in the supplemental material). The mec3
deletion strain proliferated very slowly and therefore was not tested. We found by PCR that the rad24
deletion strain was absent from our strain collection, so we did not test the effect of deleting RAD24
Rad53 is an effector kinase that causes cell cycle arrest in response to DNA damage (46
). It also may contribute to the stabilization of replication forks (34
). As with MEC1
, the deletion of RAD53
is lethal by itself but is rescued by a deletion of SML1
. We found that RAD53
was required for the viability of ORC6-rxl CDC6ΔNT
cells. In the tetrad analysis, ORC6-rxl CDC6ΔNT rad53 sml1
spores were almost always inviable (see Table S2 in the supplemental material). Deletion of CHK1
homolog) had little or no effect on the viability or proliferation rate of ORC6-rxl CDC6ΔNT
cells (see Table S2 in the supplemental material; data not shown).
The mediator protein Rad9 acts as an adaptor to couple Mec1 to Rad53. Rad9 is recruited at sites of damage and becomes phosphorylated in a Mec1- or Tel1-dependent manner (18
). The binding of Rad9 by Rad53 promotes Rad53's activating trans
autophosphorylation, which in turn promotes its dissociation from Rad9 (56
). Surprisingly, the deletion of RAD9
had a more moderate effect than the removal of MEC1
, or DDC1
on the viability or proliferation of ORC6-rxl CDC6ΔNT
cells (data not shown). One explanation for this result is that Mrc1, Rad9's functional homolog, may compensate for the loss of Rad9 in this context. This idea is supported by the decreased viability of ORC6-rxl CDC6ΔNT rad9 mrc1-AQ
spores relative to the control genotypes (see Table S2 in the supplemental material). This is consistent with a previous report, where Rad9 could compensate for the loss of Mrc1 (2
In summary, these results strongly suggest that cells with compromised rereplication controls rely on the activation of a DNA damage response requiring the sensors Mec1 and the Rad17-Ddc1-Mec3 complex and the effector kinase Rad53 for their viability. The mediator function thought to be required for coupling Mec1 to Rad53 may be provided redundantly by Mrc1 and Rad9.
Dependence on MRE11, RAD50, and XRS2.
The requirement of a DNA damage signaling pathway in ORC6-rxl CDC6ΔNT cells suggests that the cells undergo abnormally frequent DNA damage. We were curious to know what kind of DNA damage was induced in this mutant.
The evolutionarily conserved MRX complex, composed of Mre11, Rad50, and Xrs2 in yeast, is required for the signaling and repair of double-strand breaks (DSBs) (reviewed in reference 9
). The deletion of MRE11
, or XRS2
severely reduced the viability or proliferation of ORC6-rxl GAL-CDC6ΔNT-HAs
cells in plating experiments (see Fig. for MRE11
and Fig. S1 in the supplemental material for RAD50
) and of ORC6-rxl CDC6ΔNT
cells in a tetrad analysis (see Table S2 in the supplemental material). These genetic results suggest the hypothesis that double-strand breaks may occur in this genetic background. Although we do not have direct evidence for this at present, the additional genetic results presented below support this inference.
Dependence on RAD52 but not on NHEJ.
Two biochemical reactions can lead to DSB repair, namely, homologous recombination (HR) and nonhomologous end-joining (NHEJ) (28
). MRX has been reported to be required for both HR and NHEJ (reviewed in references 9
). While HR requires Rad52 and is often Rad51 dependent (49
), NHEJ requires Dnl4 and the Yku70-Yku80 complex (28
). Because we speculated that DSBs might accumulate in ORC6-rxl GAL-CDC6ΔNT-HAs
cells, we were interested to determine if one of these pathways was preferentially required in this background. Indeed, we observed that rad52
deletion was strongly lethal in the ORC6-rxl GAL-CDC6ΔNT-HAs
background (Fig. ). A weaker but reproducible synthetic lethality was obtained with RAD59
(a homolog of RAD52
), but this interaction was also observed with ORC6-rxl
alone, for unknown reasons (see Fig. S1 in the supplemental material). However, the deletion of RAD51
had no effect (see Fig. S1 in the supplemental material) (Rad52-dependent, Rad51-independent repair is reviewed in reference 49
). In contrast, the deletion of DNL4
had no effect on the slow proliferation phenotype of ORC6-rxl CDC6ΔNT
cells (see Table S2 in the supplemental material), and deletions of YKU70
had no detectable effect on the growth of ORC6-rxl GAL-CDC6ΔNT-HAs
cells on YEP-Gal (Fig. ; see Fig. S1 in the supplemental material).
These results suggest that the disruption of mechanisms preventing rereplication causes DNA lesions, perhaps DSBs, that require RAD52-dependent homologous recombination but not NHEJ.
Several additional genes known to function in maintaining genomic integrity, such as SRS2
, and CCR4
, were synthetically lethal when deleted from the ORC6-rxl GAL-CDC6ΔNT-HAs
background. In contrast, the deletion of other genes involved in DNA repair had no effect on the viability or proliferation rate of ORC6-rxl CDC6ΔNT
cells in the tetrad analysis (see Table S2 in the supplemental material). These included deletions of EXO1
(exonuclease, functions in DSB repair by recombination [20
] and the activation of Mec1 [43
(DNA helicase, required for faithful chromosome segregation) (67
), and MMS4
(endonucleases involved in DNA repair) (70
). Thus, only specific DNA damage signaling and repair pathways are vital in cells that have lost controls that prevent rereplication. In particular, we found a strong role for pathways responsible for DSB signaling and repair by HR.
Activation of Rad53.
The dependence of ORC6-rxl CDC6ΔNT cells on RAD53 suggested that Rad53 was activated in these cells. The activation of Rad53 by phosphorylation was observed using ORC6-ps,rxl GAL-CDC6ΔNT-HAs FLAG-RAD53 cells induced by the addition of galactose (Fig. ). A slower-migrating signal in an anti-Flag Western blot, presumably corresponding to phosphorylated Flag-Rad53, became visible 2 h following induction and reached a maximum between 2 and 4 h after induction. This shift in the electrophoretic mobility of Flag-Rad53 was similar to that observed when MMS (a DNA alkylating agent) was added (Fig. ). The induction of GAL-CDC6ΔNT-HAs in these cells did not cause them to rereplicate significantly, although it caused an accumulation of cells with a 2C DNA content (Fig. ). The induction of GAL-CDC6ΔNT-HAs in the presence of ORC6-ps or ORC6-rxl, but not ORC6-wt, caused a slight Rad53 activation and accumulation of 2C DNA-containing cells (Fig. ). These results suggest the speculation that the Rad53-mediated response allows ORC6 (-ps, -rxl, or -ps,rxl) GAL-CDC6ΔNT-HAs cells in YEP-Gal to pause the cell cycle during or after DNA replication, probably in order to repair some form of DNA damage (perhaps DSBs; see above) before resuming the cell cycle.
To begin to dissect the pathways involved in signaling and repairing potential DNA damage in rereplication-sensitized cells, we monitored Rad53 activation and cell cycle delays in ORC6-ps
,rxl GAL-CDC6ΔNT-HAs FLAG-RAD53
cells in which we introduced selected gene deletions. Deleting MEC1
,rxl GAL-CDC6ΔNT-HAs FLAG-RAD53
cells prevented Rad53 activation when galactose or MMS was added (Fig. ). This is consistent with the role of Mec1 in activating Rad53 (56
). The deletion of RAD17
largely, but not completely, prevented Rad53 activation, suggesting that only residual DNA damage checkpoint signaling could occur (Fig. ). Interestingly, the deletion of MRE11
,rxl GAL-CDC6ΔNT-HAs FLAG-RAD53
cells largely and reproducibly eliminated Rad53 activation induced by galactose but not by MMS (Fig. ). These results suggest that a loss of rereplication controls induces a narrower or different spectrum of DNA damage than that induced by DNA alkylation. The question of the involvement of MRX in checkpoint signaling versus a direct role in DSB repair has been controversial (9
). Our observations suggest that in the context of ORC6-ps
cells, Mre11 is required for signaling, but this does not exclude an additional function of MRX in repairing DNA damage. A requirement for Mre11 in Mec1 signaling in response to DSBs or UV has been demonstrated recently (43
), and this is consistent with our results, especially if DSBs are indeed generated in the ORC6-ps
In the absence of RAD52 (Fig. ), ORC6-ps,rxl GAL-CDC6ΔNT-HAs FLAG-RAD53 cells exhibited Rad53 activation. This places Rad52 downstream of or parallel to Rad53 and is consistent with a role for Rad52 in repairing DNA damage in cells with compromised rereplication controls and not in checkpoint signaling. Using an anti-Rad53 antiserum (a generous gift of S. Elledge), we confirmed that Rad53's gel mobility is reduced in ORC6-rxl GAL-CDC6ΔNT-HAs cells incubated in galactose (see Fig. S3 in the supplemental material). Using this assay, we found that Rad53 could still shift in the presence of deletions of CLA4, CTF4, IKI3, RTS1, SRS2, and SWI6, while a deletion of CCR4 abolished (or reduced) the Rad53 shift (see Fig. S3 in the supplemental material). Overall, our analysis places Mec1, Rad17, Ddc1, Ccr4, and Mre11 upstream of Rad53 and Rad52, Cla4, Ctf4, Iki3, Rts1, Lsm1, Srs2, and Swi6 downstream of or parallel to Rad53 in the response to a loss of mechanisms preventing rereplication.
DNA damage response limits the extent of rereplication.
We examined the effect of disrupting the DNA damage response on overt rereplication. As noted above, the detection of overt overreplication by FACS (rather than a delay with an apparent 2C DNA content) required multiple copies of GAL-CDC6ΔNT-HA
and the MCM7-NLS
,rxl MCM7-NLS GAL-CDC6ΔNT-HAm mec1 sml1
cells reproducibly showed greater rereplication than MEC1 SML1
or MEC1 sml1
controls (Fig. ; see Fig. S2 in the supplemental material). The increase in FACS signal could have been due to cells passing through mitosis without cytokinesis; if this were associated with a normal cycle of cyclin degradation, it could be expected that origins would reload normally, followed by normal reinitiation. This idea would be consistent with the checkpoint role of Mec1. However, this explanation seems unlikely, since we did not detect multinucleate cells (indicative of nuclear division without cytokinesis) or rebudded cells (indicative of mitotic exit without cytokinesis) (see Fig. S2 in the supplemental material). These results suggest that the increase in DNA overreplication in the absence of Mec1 occurred during the same S phase and was unlikely to be a gross effect due to a loss of cell cycle checkpoint function. Together, these results suggest that Mec1 restrains rereplication. We do not know why the mec1
mutants seemed to still exhibit a cell cycle arrest, but previous results indicated that the Mad2-dependent spindle checkpoint can be activated in response to DNA damage by an unknown mechanism (21
The deletion of RAD17 or MRE11 increased the extent of rereplication relative to control strains, even more than that observed with the mec1 sml1 strain (Fig. ), while the deletion of MRC1 had no or little effect (data not shown). Therefore, upstream signaling through Mec1, Rad17, and Mre11 seems to result in a restraint of rereplication.