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1 Molecular and Computational Biology Program, Department of Biological Sciences, University of Southern California, Los Angeles, California 90089-2910, USA
2 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
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Abstract |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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Because of its distribution along chromosomes, ORCs appear to be ideally positioned for involvement in other chromosomal events. Indeed, in S. cerevisiae, the ORC plays a role in the assembly of transcriptionally silent chromatin at the silent mating-type loci, HML and HMR by recruiting the silencing protein Sir1p to the HML and HMR silencers (Triolo & Sternglanz 1996; Fox et al. 1997). In Drosophila, the ORC localizes preferentially, but not exclusively, with heterochromatin and is found to associate physically with HP1, a protein involved in heterochromatin-mediated transcriptional repression (Pak et al. 1997).
In Drosophila and yeast, the ORC remains bound to chromatin throughout the cell cycle (Bell 2002). Thus, it has been suggested that ORCs may contribute to mitotic events such as chromosome condensation or segregation. Consistent with this idea, orc5ts mutants in S. cerevisiae undergo cell cycle arrest in early M phase (Dillin & Rine 1998), and orc2 and orc5 mutations in Drosophila cause defects in chromosome condensation and these cells arrest in mitosis (Landis et al. 1997; Loupart et al. 2000; Pflumm & Botchan 2001). Analysis of human cells depleted for Orc2p and Orc6p show aberrant chromosome structures and defects in mitotic progression (Prasanth et al. 2002, 2004). Nevertheless, these experiments do not resolve whether these pleiotropic defects associated with loss of ORC function reflect a direct involvement of the ORC in mitotic chromosome behavior or an indirect effect of defective chromosomal DNA replication.
To determine the cell cycle requirement for ORCs in S. cerevisiae, we have performed execution point studies. Through analysis of mutations in different subunits of the ORC, we have determined that the ORC is required during the G1/S transition for effective chromosome replication and viable progression of cells through the cell cycle. In contrast, inactivation of ORC function subsequent to replication initiation does not block replication or progression through the remainder of the cell cycle, arguing against an essential role for ORC in mitosis. We have also characterized the role of the DNA damage and spindle assembly checkpoints in response to S-phase entry with diminished ORC activity. Elimination of the spindle assembly checkpoint suppresses the proliferation defect of orcts cells under semipermissive conditions, suggesting that the spindle checkpoint is highly sensitive to diminished replication initiation.
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Results |
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To investigate the function of the ORC in the regulation of chromosomal DNA replication, we generated conditional alleles of ORC1 by PCR mutagenesis of the wild-type ORC1 gene followed by introduction into yeast cells to replace the wild-type gene. We identified a strain harboring allele orc1-161 as having excellent characteristics for cell cycle execution point analysis, including good growth at permissive temperature (23 °C) and rapid, uniform cell cycle arrest after shifting to non-permissive temperature (37 °C) (see Experimental procedures). At 23 °C, DNA content distribution of orc1-161 cells was similar to wild-type cells, except for a reduced proportion of cells in G1 (Fig. 1A, Time = 0). Shift to 37 °C of logarithmically growing orc1-161 mutant cells resulted in: cell cycle delay with severely limited chromosome replication (Fig. 1A), rapid loss of viability (Fig. 1B), and cell cycle arrest with pre-anaphase spindles and large-budded morphology (see below). These characteristics indicate that the orc1-161 mutant strain is defective in chromosomal DNA replication resulting in pre-anaphase cell cycle arrest.
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To examine the role of ORC1 in different cell cycle stages, we analyzed DNA replication and cellular viability after eliminating ORC1 function at specific points in the cell cycle. First, we tested the requirement of ORC1 during late G1 and early S-phase. Wild-type and orc1-161 cells grown at 23 °C were arrested in late G1-phase by 
-factor treatment or G1-cyclin depletion, shifted to 37 °C for 1 h to inactivate Orc1-161p, and then released from the G1 block at 37 °C. Whereas wild-type cells completed DNA replication 
1 h after release, orc1-161 cells required over 3 h to reach a fully replicated DNA content (Fig. 2A). The orc1-161 strain lost viability concurrent with entry into S-phase (Fig. 2B,C), and this lethality was not rescued by inhibition of DNA synthesis with hydroxyurea (HU) (Fig. 2C). Thus, once cells have attempted S-phase entry with diminished ORC1 function, restoration of ORC1 activity (by plating cells at 23 °C) does not rectify the DNA replication defect, even if replication elongation is delayed. This is not due to an inability to rapidly restore Orc1-161p activity upon return to the permissive temperature, because viability was maintained when G1-blocked cells incubated at 37 °C were returned to the permissive temperature preceding their release into S-phase (see below). Thus, ORC1 function is required during the late G1 to early S-phase period for normal kinetics of chromosomal DNA replication, consistent with a role for ORC1 in replication initiation.
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1 h following release from HU (Fig. 3A,B). In contrast, cells harboring a temperature-sensitive allele of DNA Polymerase (cdc17-1) failed to complete replication following release from the early S-phase block, consistent with the replication elongation function of this enzyme (Fig. 3A). Wild-type and orc1-161 cells also completed mitosis and cytokinesis with very similar kinetics, based on the accumulation of cells with G1 DNA content, which began 2 h after release (Fig. 3A,B, -factor was added back in (A) to enable detection of cells in G1-phase). As expected, when the orc1-161 cells were allowed to proceed to the second S-phase (no -factor), defective replication was observed as cells accumulated with incompletely replicated chromosomes (Fig. 3B).
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It was possible that the defective chromosomal replication and loss of viability that occurred upon initiating the second cell cycle was due to lack of an ORC1 function executed during the preceding cycle. Therefore, we delayed entry of HU block-and-released cells into the subsequent S-phase by arresting them in G1 and restoring ORC1 function by plating cells at 23 °C. Because incomplete G1-arrest was attained with the -factor at 37 °C (based on budding morphology, data not shown), we utilized strains with the G1 cyclin CLN2 under control of the methionine-repressible MET promoter and the native CLN1-3 genes deleted. Addition of methionine, after early S-phase block-and-release at 37 °C, blocked cells in the succeeding G1-phase and prevented the loss of viability of orc1-161 cells (Fig. 3E). This result demonstrates the reversibility of the orc1-161 defect in late G1-phase, even after passage through late S, G2, M, and early G1 at 37 °C. Taken together, these results indicate that Orc1p is able to execute its essential function(s) within the late G1 to early S period to promote replication initiation. Following initiation, Orc1p is not essential for the completion of subsequent cell cycle processes such as replication elongation, mitosis, and cytokinesis.
ORC2 and ORC5 function is required only during the G1 to early S period
We wished to extend our analysis to the role of the entire ORC complex and further explore the possibility that the ORC has cell cycle functions in addition to replication initiation. Therefore, we performed similar experiments with temperature-sensitive alleles of two other ORC subunits, ORC2 (orc2-1) and ORC5 (orc5-1) (Foss et al. 1993; Loo et al. 1995). Incubation of unsynchronized orc2-1 and orc5-1 cells at 37 °C resulted in accumulation of 2C DNA content in these cells (Fig. 1A). Although this result might suggest a role for Orc2p and Orc5p in a cell cycle event subsequent to replication initiation, it might instead reflect less severe depletion of ORC function by these conditional alleles in comparison with the orc1-161 allele. Consistent with this interpretation, orc1-161 cells accumulated 2C DNA content when incubated at 30 °C (data not shown).
Similarly to orc1-161 cells, orc2-1 and orc5-1 cells replicated poorly after G1 block-and-release at 37 °C, with accompanying loss of viability (Fig. 2A,B). Also similarly to the orc1-161 cells, orc2-1 and orc5-1 cells were delayed in cell division, consistent with checkpoint activation in response to incomplete DNA replication (Fig. 2A,B). Some leakiness of the orc5-1 allele is apparent, as a fraction of orc5-1 cells completed replication and cell division, beginning at 2 and 2.5 h, respectively. Nevertheless, this was a significant delay compared with wild-type cells, and a substantial proportion of orc5-1 cells failed to undergo cell division even after 4 h. Thus, Orc2p and Orc5p are each required during late G1 to early S for normal kinetics of DNA replication.
After release at 37 °C from early S-phase, orc2-1 and orc5-1 mutants replicated with kinetics similar to the wild-type strain (Fig. 3A,B). Defective replication was not observed until the second S-phase, when cells accumulated with incompletely replicated chromosomes (Fig. 3B, 3 and 4h). The timing of accumulation of cells re-blocked in G1 with -factor following early S block-and-release indicated that the timing of cell division in the orc2-1 and orc5-1 strains was similar to wild-type (Fig. 3A). Consistent with this conclusion, spindle elongation occurred with comparable timing in the orc2-1, orc5-1 and wild-type strains after early S block-and-release (Fig. 3C). The viability loss of the orc2-1 and orc5-1 strains exhibited similar kinetics as the orc1-161 strain, with minimal loss of viability until passage through the G1/S transition of the next cell cycle (Fig. 3D). All together, these results indicate that ORC1, ORC2 and ORC5 are essential during late G1 to early S-phase to promote the replication initiation, after which, ORC1, ORC2 and ORC5 are not required for later events such as replication elongation, mitosis, and cytokinesis.
ORC1, ORC2 and ORC5 loss of function eliminates ORC-origin DNA binding
Previously, using ChIP, we showed that the orc1-161 and orc2-1 mutations result in a loss of ORC's ability to bind specifically to replication origins at 37 °C in G2/M-arrested cells (Aparicio et al. 1997) (O.M.A and S.P.B., unpublished observations). In orc5-1 mutant cells, integrity of the ORC is lost at 37 °C (Loo et al. 1995). In the case of orc1-161, immunoprecipitation of the ORC (using anti-Orc3 or anti-Orc2 monoclonal antibody) showed some reduction in the level of intact ORC at 23 °C; however, shift to 37 °C resulted in loss of Orc1-161p without affecting the level or integrity of the remainder of the complex (Fig. 1C). In vitro analysis of partial ORCs indicates a requirement of intact ORC for origin-DNA binding (only Orc6p appears dispensable) (Lee & Bell 1997), consistent with the results of the ChIP assay in vivo. All of these results argue that ORC-origin binding is strongly reduced or eliminated in orc1-161, orc2-1, and orc5-1 cells at the non-permissive temperature.
To examine ORC-origin DNA binding throughout the cell cycle in the orc1-161, orc2-1 and orc5-1 strains, we analyzed the chromatin association of the ORC with origin (ARS305 and ARS607) and non-origin DNA (305+8kb and 607+15kb) following early S-phase block-and-release at 37 °C using ChIP with an anti-ORC polyclonal antibody that recognizes all six ORC subunits. In each of the mutant strains, ORC-origin association was reduced to non-origin levels upon thermal inactivation of the Orcts protein indicating loss of the entire ORC from the chromatin (Fig. 4). In contrast to the orcts strains, ORC-origin binding remained robust in wild-type cells. Because inactivation of Orc1-161p, Orc2-1p and Orc5-1p resulted in loss of ORC-origin DNA binding, and because replication elongation, mitosis, and cell division occurred with normal kinetics and minimal loss of cell viability in each of the mutant strains, our data strongly support the conclusion that ORC-origin DNA binding is dispensable for these postinitiation processes.
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Defective replication can lead to activation of the DNA replication and/or DNA damage checkpoints resulting in induction of DNA metabolism genes, inhibition of late-firing replication origins, reduction in the overall rate of DNA synthesis, and delay of mitosis (reviewed in Bartek et al. 2004). Inactivation of Orc1-161p at 37 °C caused incomplete chromosomal DNA replication and delayed cycle progression, consistent with checkpoint activation (Figs 1A and 2A). Similar experiments with orc1-161 cells shifted to 33 °C gave similar results (this temperature yielded clearer and more consistent results in the subsequent series of experiments), including incomplete DNA replication (Fig. 5A,B) and accumulation of large-budded cells with preanaphase spindles (Fig. S1). Furthermore, the level of Clb2p, which normally is degraded in late mitosis, declined at 1.5 h in wild-type cells, but remained elevated in orc1-161 cells (Fig. 5C). These phenotypes are consistent with checkpoint restraint of mitosis.
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Rad53p activation occurs through distinct signaling pathways that sense either the presence of DNA damage or replication stress. Rad9p is required to transduce DNA damage signals to activate Rad53p (Bartek et al. 2004). To determine whether Rad53p activation resulting from loss of ORC1 function reflected the presence of DNA damage, we analyzed Rad53p phosphorylation in orc1-161 cells lacking RAD9. Rad53p activation was eliminated in orc1-161 rad9
cells, consistent with DNA damage occurring in cells that attempt replication with diminished ORC1 function (Fig. 5D).
As an additional test for the presence of DNA damage in cells with reduced ORC1 function, we analyzed the subcellular localization of Ddc2p, which is recruited to sites of DNA damage (Melo et al. 2001; Rouse & Jackson 2002). In cells expressing Ddc2p fused to green fluorescent protein (Ddc2-GFP), DNA damage causes the accumulation of Ddc2-GFP into one or more fluorescent foci. In wild-type cells at 33 °C, Ddc2-GFP was present throughout the nucleus and did not accumulate into foci (Fig. 5E). However, in orc1-161 mutants at the non-permissive temperature, at least one Ddc2-GFP focus was observed in 99% of the cells and these cells had an average of nine foci per cell, consistent with the presence of DNA damage in these cells (Fig. 5E).
We also analyzed phosphorylation of Pds1p, which occurs in response to DNA damage but not in response to replication inhibition caused by HU (Cohen-Fix & Koshland 1997). Pds1p phosphorylation was first detected 1 h following G1 release and persisted for at least 5 h (Fig. 5F). Together with the RAD9 dependence of Rad53p phosphorylation and the presence of Ddc2p foci, these results indicate activation of the DNA damage checkpoint and suggest that DNA damage occurs in cells defective in ORC1.
Cell cycle arrest of orc1-161 cells is not due solely to the DNA damage checkpoint
Inactivation of ORC1 function at 33 °C (or above) results in cell cycle arrest with characteristics of a DNA damage-dependent mitotic checkpoint. To directly analyze the involvement of the DNA damage and/or replication checkpoints in the mitotic delay of orc1-161 cells, we tested the effects of mutations in RAD53 and CHK1, which mediate mitotic delay through parallel, MEC1-dependent signaling pathways (Bartek et al. 2004). In unsynchronized cultures shifted to 33 °C, deletion of CHK1 did not significantly alter the DNA content distribution of orc1-161 cells until 5–6 h when a small fraction of cells with 1C DNA content emerged, suggesting the mitotic delay remained largely intact (Fig. 5A). Interestingly, orc1-161 rad53-11 and orc1-161 rad53-11 chk1 cells accumulated a greater DNA content than orc1-161 cells (Fig. 5A), suggesting that Rad53p reduces the level of DNA synthesis in orc1-161 cells at the non-permissive temperature, perhaps through the intra-S checkpoint (Bartek et al. 2004). Regardless, DNA content distribution suggested the mitotic delay remained largely intact in orc1-161 rad53-11 and orc1-161 rad53-11 chk1 cells. In support of this, Clb2p levels remained elevated in orc1-161 rad53-11 cells for at least 4 h and the majority of cells remained large-budded (Fig. 5C and data not shown). Thus, mitotic arrest of Orc1p-deficient cells is not due solely to Rad53p- and Chk1p-mediated cell cycle arrest.
Loss of ORC1 function activates the spindle assembly checkpoint
The G2/M accumulation of orc1-161 rad53-11 chk1 cells suggests that an additional mechanism restrains mitotic progression of cells lacking ORC1 function. The spindle attachment checkpoint delays the onset of anaphase until the kinetochore of each sister chromatid is properly attached to the mitotic spindle (reviewed in Lew & Burke 2003). We considered that defective ORC1 function might result in incomplete replication or damage of centromeric DNA leading to improper kinetochore assembly at centromeres, and hence, spindle checkpoint activation. To carefully monitor the number of cell division events undertaken by orc1-161 cells and checkpoint-defective derivatives of these cells, we analyzed microcolony formation at 30 °C, which is semipermissive for DNA replication of orc1-161 cells resulting in accumulation with 2C DNA content (data not shown). We dissected individual, small-budded cells on to prewarmed plates of rich medium and counted cells after 24 h. Small-budded cells had already initiated S-phase, so these cells were expected to complete the first cell division normally.
The orc1-161 cells formed microcolonies with an average of 44 cells, indicating that these cells divided about 4 times following the initial cell division (Fig. 6A). The orc1-161 rad53-11 cells formed microcolonies with an average of 97 cells, indicating that these cells underwent one extra round of cell division, presumably due to loss of the checkpoint. Remarkably, deletion of MAD2, a required component of the spindle assembly checkpoint, enabled orc1-161 cells to undergo about five additional cell division cycles. The ability of orc1-161 mad2 cells to complete these extra cell divisions depends on the DNA damage checkpoint, as introduction of the rad53-11 mutation into these cells eliminated the extra cell division cycles, reducing the average number of cells per microcolony to 22. Thus, orc1-161 rad53-11 mad2 cells completed one fewer cell division cycle than orc1-161 cells, two fewer than orc1-161 rad53-11 cells and six fewer than orc1-161 mad2 cells. This result indicates that the presence of at least one checkpoint enhances the survival of orc1-161 cells. Together, these results strongly suggest that the spindle checkpoint responds to defects resulting from lack of ORC1 function and that the DNA damage and spindle assembly checkpoints play distinct, but overlapping, roles in sustaining the viability of orc1-161 cells. Whereas the spindle checkpoint appears to play a larger role in delaying cell division, the DNA damage checkpoint becomes critical in preventing lethality in cells lacking the spindle checkpoint.
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were inviable. Deletion of MAD2 did not restore the viability of orc1-161 cells at 33 °C that fail to complete DNA synthesis (Fig. S2 and data not shown). Together with the microcolony assay, these findings show that MAD2 restrains cell division in cells with reduced ORC1 function. The ability of the orc1-161 mad2 cells to proliferate also indicates that the orc1-161 defect(s) is not necessarily lethal at 30 °C providing the DNA damage checkpoint remains functional. However, loss of the spindle checkpoint might allow proliferation of cells with sublethal defects in chromosome segregation, which are not apparent in our assay. To confirm that the effect of deleting MAD2 reflected its role in the spindle attachment checkpoint, we tested the effect of deleting another component of this checkpoint, BUB1. Deletion of BUB1 partially rescued the viability of orc1-161 cells, consistent with a role for the spindle attachment checkpoint in cells with defective ORC1 function (Fig. 6C). We also tested the effect of deleting BUB2, an essential component of the spindle position checkpoint, which monitors the orientation of the mitotic spindle along the mother-bud axis, but is not required for full function of the spindle assembly checkpoint (Lew & Burke 2003). In contrast to deletion of MAD2 and BUB1, BUB2 deletion did not suppress the lethality of orc1-161 cells (Fig. 6C), suggesting that ORC1 inactivation does not alter spindle position. Activation of the spindle checkpoint appears to be a conserved response to replication defects, as deletion of MAD2 also rescued the viability of orc2-1 and cdc17-1 cells (Fig. 6D).
The DNA damage and spindle assembly checkpoints independently restrain mitosis of replication defective cells
The RAD53 requirement for the viability of orc1-161 mad2 cells may reflect its function in mitotic delay and/or in DNA repair. To understand better how RAD53 maintained the viability of orc1-161 mad2 cells, we compared the DNA content of these cells with that of orc1-161 mad2 rad53-11 cells. In unsynchronized cultures shifted to 33 °C, orc1-161 mad2 strains accumulated with partially replicated DNA content (Fig. 7A), similar to orc1-161 rad53-11 cells (Fig. 5A). In orc1-161 cells lacking both RAD53 and MAD2 checkpoint functions, a small proportion of cells with 
1C DNA content began to emerge after about 3 h (Fig. 7A). DNA content < 1C suggests that these cells have aberrantly segregated chromosomes, which can explain their lethality. Thus, RAD53 and MAD2 independently contribute to mitotic arrest in cells lacking ORC1 function. CHK1 also plays a role in mitotic delay in ORC1-deficient cells, as deletion of CHK1 in orc1-161 mad2 and orc1-161 mad2 rad53-11 cells further increased the proportion of cells that divided (Fig. 7A). These results show that CHK1, RAD53 and MAD2 act in parallel pathways to restrain cell division in ORC1-deficient cells.
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To determine whether dual checkpoint activation is a general response to replication initiation defects, we examined the effect of eliminating these checkpoint pathways in strains with a conditional mutation of CDC6 (cdc6-1). Similar to inactivation of Orc1-161p, inactivation of Cdc6-1p at 33 °C resulted in accumulation of cells with incompletely replicated chromosomes (Fig. 7C). Introduction of the rad53-11 or mad2 mutation into the cdc6-1 cells allowed a small proportion of these cells to escape the mitotic arrest with incompletely replicated chromosomes, resulting in the emergence of some cells with 1C DNA content (Fig. 7C). Elimination of both checkpoint proteins resulted in a significant increase in the proportion of these cells, indicating that these cells have escaped the mitotic arrest and improperly segregated chromosomes (Fig. 7C). This result suggests that activation of both the DNA damage and spindle assembly checkpoints is a conserved response to a replication initiation defect.
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Discussion |
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This study aimed to gain a more thorough understanding of the function of the ORC during the eukaryotic chromosome replication and segregation cycle. We have demonstrated that depletion of ORC1, ORC2 or ORC5 function in G1-arrested cells rapidly and severely impedes chromosomal replication and production of viable daughter cells (Fig. 2). We have shown previously that inactivation of ORC1 or ORC2 in G1-arrested cells reduces MCM-origin DNA association, suggesting that the ORC is required to maintain pre-RCs during G1-phase in S. cerevisiae (Aparicio et al. 1997). Hence, loss of pre-RC integrity due to ORC inactivation can explain the replication defect these cells exhibit upon S-phase entry. Our findings do not exclude an additional, unrecognized role of the ORC in replication initiation. ORC-defective cells also lose viability coincident with S-phase entry, and delaying replication elongation with HU does not rescue the viability of these cells (Fig. 2C). Thus, once these cells have progressed far enough into S-phase for Clb-kinase levels to become inhibitory for pre-RC assembly, probably coincident with the "point of no return" defined for Cdc6p (Piatti et al. 1996), return of these cells to permissive conditions is futile and DNA replication and viability cannot be restored. These cells are committed to a cell cycle lacking pre-RC directed replication.
Our results also imply that pre-RCs can be established during late G1-phase, because return of orc1-161 cells to permissive conditions prior to release from a late G1 arrest prevents the lethality and defective replication due to S-phase entry in the absence of ORC function (Fig. 3E and data not shown). This finding probably explains why our results did not demonstrate a requirement of ORC function in early G1, when pre-RC assembly is thought normally to occur. However, pre-RC assembly in late G1 also may be normal. For example, late G1 transcription of CDC6 is important for effective replication in small daughter cells, suggesting that assembly of at least a subset of pre-RCs occurs in late G1 in many if not all cells (Piatti et al. 1995). In addition, the potential for pre-RCs to assemble in late G1 is crucial for stationary phase cells re-entering the cell cycle, as pre-RCs are disassembled in stationary phase (Diffley et al. 1994).
Our results suggest that the ORC is required for the maintenance of pre-RCs during G1. However, previous studies in yeast and in Xenopus egg extracts have suggested that the ORC is not required to maintain MCM-chromatin association following pre-RC assembly (Hua & Newport 1998; Rowles et al. 1999; Shimada et al. 2002). In the earlier yeast study, depletion of Orc2-1p in G1-arrested cells did not affect MCM-chromatin association or subsequent S-phase progression (Shimada et al. 2002). In contrast, we clearly observed effects on replication and/or MCM-chromatin association with mutations in ORC1, ORC2, and ORC5 (Fig. 2) (Aparicio et al. 1997). The reason for this difference is unclear, but may reflect the higher restrictive temperature used in our experiments, which might more effectively destabilize Orc2-1p-containing pre-RCs. Our observations may reflect a requirement to re-establish pre-RCs in yeast cells undergoing an extended G1 period, such as that caused by -factor arrest. Perhaps local chromatin dynamics such as transcription destabilize the pre-RC. If so, some pre-RCs may be significantly more sensitive to ORC depletion depending on their location in relation to transcription units. Consistent with this, plasmids with different Autonomously Replicating Sequences (ARS) are propagated with significantly different efficiencies in mcmts cells under semipermissive conditions, an effect which is directly related to the local transcriptional environment surrounding the ARS (Maine et al. 1984; Nieduszynski et al. 2005). In addition, high level transcription through a replication origin can abolish its activity (Snyder et al. 1988). In contrast, Xenopus egg extracts are devoid of transcription, and the onset of transcription later in development coincides with more stringent specification of replication origin location within the chromatin (Hyrien et al. 1995). Thus, the requirement for ORC function in the maintenance of pre-RCs may depend on the cell type, proliferation stage, and/or local transcriptional context.
ORC is dispensable following initiation of chromosomal replication
Our results show that ORC performs no essential role in the S. cerevisiae cell cycle subsequent to the initiation of DNA replication. After early S-phase block-and-release, ORC is not required for the timely completion of replication elongation, mitosis or cytokinesis, and viability remained high (until the next G1/S) (Fig. 3). This is observed in three different ORC mutant strains exhibiting a null phenotype for ORC-origin binding (Fig. 4). Therefore, we infer that all functions of ORC that depend upon its binding to origin DNA are defective in these strains. Hence, if ORC (or an individual Orc protein) does perform an essential role in postinitiation cell cycle events, this function must be independent of origin-DNA binding by ORC and independent of fully intact ORC, because the orc1-161 and orc5-1 mutations also result in loss of ORC integrity (Fig. 1C) (Loo et al. 1995). It is possible that, in addition to replication initiation, ORC carries out an event required for mitosis (or other late cell cycle event) that is executed early in the cell cycle (prior to or during the early S-phase block) and does not depend on continued presence of ORC (subsequent to the early S-phase block). Participating in the initiation of orderly chromatin assembly or the establishment of sister chromatid cohesion during replication could represent such a function.
A previous report concluded that S. cerevisiae ORC has an essential role in mitosis based on analysis of the orc5-1 (and a collection of orc5ts) strain (Dillin & Rine 1998). The key results were that orc5-1 cells exhibited cell cycle arrest with G2/M DNA content and cell morphology, a high (preanaphase) level of cyclin-dependent kinase activity, and the G2/M arrest was not due to the DNA damage and/or replication checkpoints. Our findings, however, show that the spindle assembly checkpoint also must be disabled to eliminate the mitotic arrest (Fig. 7). This result is consistent with the mitotic arrest of orc5ts cells resulting from inefficient replication initiation, rather than a role of ORC in early mitosis. Furthermore, our experiments showing that cells deficient in ORC1, ORC2 and ORC5 proceed normally through late S, G2 and M phases in the absence of detectable ORC-origin DNA binding provide strong evidence that ORC is not required to execute an essential function during these cell cycle stages.
Decreased ORC function results in a DNA damage response
Replication initiation with deficient ORC1 function results in Rad9-dependent activation of Rad53, Ddc2 focus formation, and Pds1p phosphorylation, indicating the presence of damaged DNA (Fig. 5). Previous studies also have suggested the presence of DNA damage due to loss of ORC function (Garber & Rine 2002; Watanabe et al. 2002). The mechanism through which initiation defects result in DNA damage is unclear. One possibility is simply that ORC deficiency result in fewer, normal initiation events. As a result, the average replicon will be longer, which may increase the frequency of replication fork collapse. Collapsed replication forks are likely to be recognized as damaged DNA due to the presence of single-stranded DNA and/or free DNA ends. However, there is no direct evidence that the likelihood of fork collapse increases with increasing replicon size due to limited processivity of the replisome per se. Nevertheless, an inevitable result of increased replicon size is a corresponding increase in the number of replication fork pause sites or damaged DNA template sites that each fork will encounter, along with a decreasing likelihood of rescue by a converging replication fork due to decreased initiation of flanking origins.
In support of the idea that decreased initiation alone is sufficient to create these conditions, we have recently shown that DNA damage occurs in clb5 cells, which are deficient in S-phase CDK activity (Gibson et al. 2004). In these cells, reduced initiation, particularly of late-firing origins, increases average replicon size, similar to the situation in ORC-deficient cells (Donaldson et al. 1998). Interestingly, in both clb5 and orc1-161 mutants, Rad53 phosphorylation occurs in late S-phase, suggesting the accumulation of DNA damage as replication proceeds (Fig. 5) (Gibson et al. 2004). Alternatively, DNA damage may occur early in S-phase, but fail to elicit robust Rad53 activation if the threshold for Rad9-dependent signaling is higher during S-phase (Navas et al. 1996; Shimada et al. 2002). A significant difference between the ORC-deficient and Clb5-deficient cells, however, is that Clb5-deficient cells experience only a transient DNA damage response and cell cycle delay, which probably reflects the greater levels of initiation in these cells (Gibson et al. 2004).
An alternative, but not exclusive, explanation for the DNA damage response in ORC-deficient cells is improper or incomplete pre-RC assembly or activation. Although evidence is lacking, it is possible that at some origins only partial pre-RC assembly occurs due to instability of the ORC-origin DNA binding. It is thought that at least two MCM complexes assemble at each pre-RC, one acting as the replicative helicase for each replisome (reviewed in Takahashi et al. 2005). The association of a single MCM complex at a replication origin might result in assembly of a single, unidirectional replisome. While the single replisome might function normally, DNA ends might remain exposed at the origin and elicit a DNA damage response.
Decreased ORC function activates the spindle checkpoint
Our findings show involvement of the spindle assembly checkpoint in the response to replication initiation defects, and similar findings have been published since the inception of this study (Stern & Murray 2001; Garber & Rine 2002). This checkpoint regulates the metaphase to anaphase transition by monitoring bipolar attachment of spindle microtubules to each sister kinetochore by sensing tension at kinetochores, which results from spindle extension counteracted by sister chromatid cohesion (reviewed in Lew & Burke 2003). Satisfaction of this checkpoint requires duplication of all centromeres, proper kinetochore assembly at each duplicated centromere, and the establishment of cohesion between all sister chromatid pairs. The partial replication and DNA damage in orc1 mutant cells may disrupt kinetochore function and/or the establishment of sister chromatid cohesion. We have been unable to determine whether cohesion is defective in orc1 mutant cells (perhaps due to the replication defect itself). The exact mechanism responsible for activation of the spindle assembly checkpoint in these cells remains unclear.
ORC plays an indispensable role in the initiation of eukaryotic chromosomal DNA replication. The critical nature of this process has led to the evolution of multiple overlapping mechanisms to ensure its accuracy and completion, as well as its intimate coordination with the segregation machinery. Our demonstration that budding yeast with diminished ORC1 function activate seemingly independent cell cycle checkpoint pathways suggest that defective replication initiation creates multiple defects in the structure of chromosomes and that the surveillance mechanisms are highly sensitive to these perturbations. The overlapping sensitivities to chromosomal defects of these checkpoint pathways may help prevent the proliferation of damaged cells that might otherwise escape detection. Indeed, the continued proliferation of orc1-161 mad2 suggests that the spindle assembly checkpoint reinforces the first line of defense imposed by the DNA checkpoint pathways. In higher cells, a more robust cell cycle delay by the combined action of these checkpoints may help prevent cancer by more securely blocking proliferation of cells with severely damaged chromosomes and channeling these cells into apoptosis.
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Experimental procedures |
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Strains are derived from W303 and described in Table 1. Most gene deletions and epitope tags were constructed using long oligonucleotide-mediated transformation with the HIS5 selectable marker. MAD2 was deleted using plasmid pmad2-URA3 (from P. Sorger). RAD53-HA3 was introduced with plasmid p306-RAD53-HA. Constructs were confirmed by PCR and/or immunoblotting. Construction of ORC1 temperature-sensitive strains is as follows. We constructed a "swapper strain" harboring a chromosomal deletion of ORC1 complemented by the wild-type ORC1 gene in pRS316 (CEN/URA3). A 1207 bp region of ORC1, which contains the ATP binding and hydrolysis domain and shares extensive sequence similarity with CDC6, was amplified by PCR under mutagenic conditions (Leung et al. 1989). The amplified ORC1 segments were ligated into an integrating vector (pRS405) containing the wild-type ORC1 gene with the corresponding region of ORC1 removed. Following transformation into E. coli, approximately 8000 individual clones were harvested together as a "library" for DNA preparation. The plasmid DNA library was digested for integration into the chromosomal leu2 locus, and introduced into the swapper strain by transformation; LEU+ transformants were selected at 23 °C. After being picked on to medium lacking leucine and allowed to grow at 23 °C, approximately 5400 LEU+ clones were transferred to plates containing 5-fluoro-orotic acid (5-FOA) to select for cells that had lost the URA3 plasmid carrying the wild-type ORC1 gene. Cells resistant to 5-FOA, which therefore harbored newly introduced alleles of ORC1 that retained at least partial function at 23 °C, were tested for the ability to grow at 37 °C by replica-plating on to rich medium. Temperature-sensitive candidates were re-tested by streaking for single colonies on rich medium at 37 °C. These strains were complemented for growth at 37 °C by wild-type ORC1 on a low-copy (CEN) plasmid indicating that these strains each harbored a recessive, temperature-sensitive mutation in ORC1. To identify mutant strains that exhibited rapid and uniform cell cycle arrest after shifting to 37 °C, we selected 35 mutant strains and microdissected logarithmically growing cells on solid, prewarmed, rich medium and identified seven strains that arrested growth as small microcolonies with a predominance (> 90%) of four or fewer cells/buds even after 12–18 h at 37 °C (data not shown). Based on the results of this analysis and the DNA content distribution of these strains after temperature shift, we selected the strain harboring allele orc1-161 for further study.
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DNA content analysis and -factor block-and-release have been previously described, except that cells were shifted to 37 °C or 33 °C for 1 h prior to release from -factor (Aparicio et al. 2004). In some experiments, to minimize any potential residual protein function, we performed the thermal shift for 2 h (e.g. Fig. 3); however, we observed no significant difference relative to a 1 h temperature shift. Cells were blocked in early S-phase by incubation with 200 mM hydroxyurea for 3 h at 23 °C. Chromatin immunoprecipitation (ChIP) has been described (Aparicio 1999). For viability determination, cells were sonicated and plated at 23 °C in duplicate on to YEPD or medium lacking methionine where indicated. After three days, colonies were counted, and the average was plotted relative to the Time = 0 value. One representative from multiple experimental sets is shown. Microdissections were performed with a Singer MSM200.
Protein analyses
Proteins were extracted by trichloroacetic acid (TCA) precipitation, except for immunoprecipitation with anti-Orc3 antibody (SB3). Anti-Ha 16B12, anti-Myc (9E10), and anti-Clb2 (Santa Cruz Biotechnology) antibodies were followed with HRP-conjugated anti-mouse or anti-rabbit secondary antibody (GE Healthcare). HRP was detected using Super Signal Elisa Femto (Pierce), and analyzed using a Bio-Rad ChemiDoc system and QuantityOne software.
Microscopy
Spindles were visualized with rat anti-tubulin (Serotec) and FITC-conjugated anti-rat (Zymed) as described (Gibson et al. 2004). GFP fixation is described at <http://www.ciwemb.edu/labs/koshland/Protocols/MICROSCOPY/gfpfix.html>. GFP was detected using an Olympus IX71 microscope with 100X, 1.4 NA PlanApo oil immersion objective and Chroma FITC filter set (ex. 485/20 nm, em. 515/30 nm). Images were captured with a Roper Scientific DV42059 camera and visualized using SoftWoRx (Applied Precision). At least 100 cells were photographed in several focal planes through the nuclei to quantify foci under constant conditions. Quantifications of cell and spindle morphologies were determined by analysis of at least 200 cells at each time point.
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Acknowledgements |
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* Correspondence: E-mail: oaparici{at}usc.edu
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