A Simplified Microarray CGH Assay for DNA Replication
We have adapted and streamlined existing microarray assays (Raghuraman et al., 2001
; Yabuki et al., 2002
) to create a rapid and economical genome-wide assay for yeast DNA replication. Our simplified assay uses CGH to directly measure the increase in DNA copy number arising from replication or re-replication. During S phase replication, the copy number of each DNA segment reflects the timing of its replication because the earlier a DNA segment replicates, the greater the proportion of replicating cells containing a duplication of this segment. Origins, which replicate earlier than neighboring regions, can be localized to chromosomal segments where the copy number reaches a local maxima. Thus, use of microarray CGH to monitor copy number changes across the genome can provide a comprehensive view of the location and efficiency/timing of initiation sites during replication and re-replication.
shows a schematic of our microarray CGH replication assay. Genomic DNA from replicating (or rereplicating) and nonreplicating cells is purified and differentially labeled with Cy5 and Cy3. The labeled probes are competitively hybridized to a spotted microarray and the raw Cy5/Cy3 values are normalized such that the average ratio corresponds to the DNA content determined by flow cytometry. Data are smoothed and origins are computationally identified by locating prominent and reproducible peaks in smoothed replication profiles.
Figure 1. Use of comparative genomic hybridization (CGH) on spotted microarrays to assay DNA replication. (A) Schematic representation of the CGH replication assay. Genomic DNA is purified from nonreplicating and replicating cells, differentially labeled with Cy3 (more ...)
Before using the microarray CGH assay to study re-replication, we assessed its reproducibility and its ability to identify known replication origins in the S phase of a wild-type S288c strain (flow cytometry data in ). and Supplementary Figure S2 show the mean of the smoothed S phase replication profiles from four hybridizations plus or minus the “experiment variability” (see Materials and Methods
) for chromosome X. The small variability demonstrates that this technique is highly reproducible. An overlay of our replication profiles with those generated from previously published data (Raghuraman et al., 2001
; Yabuki et al., 2002
) shows considerable agreement in both peak positions, which reflects origin locations, and peak heights, which reflects origin timing/efficiency. When our peak finding algorithm was applied to our profiles, we obtained origin numbers (212) comparable to those obtained by Rhaguraman et al.
(2001) (332) and Yabuki et al.
) (260). Additionally, the alignment of peaks to origins systematically mapped by 2-D gel electrophoresis or ARS plasmid assay was similar to, or better than, published data (Supplementary Table S1). Together, these data confirm that our streamlined assay is reproducible and accurate.
Re-replication Competent Mutant Has a Mostly Normal S Phase
We have previously demonstrated that simultaneous deregulation of three pre-RC components (ORC, Mcm2-7, and Cdc6) leads to limited re-replication in G2/M phase arrested cells (Nguyen et al., 2001
). These initiation proteins were deregulated by mutations that make the proteins refractory to CDK regulation. First, the CDK consensus phosphorylation sites of two subunits of the origin recognition complex, Orc2 and Orc6, were mutated, preventing Cdc28/Cdk1 phosphorylation of these subunits (orc2-cdk6A, orc6-cdk4A
). Second, two copies of the SV40 nuclear localization signal were fused to MCM7
) to prevent the Cdc28/Cdk1 promoted net nuclear export of the Mcm2-7 complexes. Finally, an extra copy of CDC6
, containing a partially stabilizing N-terminal deletion, was placed under control of the galactose inducible promoter (pGAL1-
). This strain re-replicates when Δntcdc6 is induced by addition of galactose and will be referred to as the OMC re-replicating strain in reference to its deregulation of O
cm2-7, and C
A major concern in any genetic analysis of replication control is the possibility that the mutations deregulating replication proteins also disrupt their replication activity. Such a nonspecific perturbation would complicate any interpretation of the resulting phenotype. We and others have previously reported that Δnt-cdc6 expressed under the CDC6
promoter retains full replication initiation function (Drury et al., 2000
; Nguyen et al., 2001
). To determine whether the mutations deregulating Orc2, Orc6, and Mcm7 in the OMC strain also preserve their initiation function, we compared S phase of the OMC strain (orc2-cdk6A orc6-cdk4A MCM7-2NLS pGAL1-
), when re-replication was not induced, to S phase of the congenic wild-type A364a strain (ORC2 ORC6 MCM7 pGAL1
). When cells were harvested at the same point in S phase (), the replication profiles for the two strains showed considerable overlap (, Supplementary Figures S3 and S4), although ORC and Mcm7 mutations cause subtle alterations in the initiation of DNA replication. Because two wild-type strains of different strain backgrounds show nearly identical replication profiles (Supplementary Figures S5 and S6), we believe these differences reflect subtle alterations in the initiation activity of the mutant ORC and Mcm2-7. Nonetheless, we conclude that, overall, the mutant ORC and Mcm2-7 proteins in the OMC strain retain most of their normal initiation activity.
Mapping Reinitiating Origins
A key prediction of the current model for eukaryotic replication control is that pre-RC reassembly and reinitiation should only occur where pre-RCs normally assemble, i.e., the potential origins or pro-ARSs identified by Wyrick et al.
). In our previous characterization of re-replication induced at G2/M phase in the OMC strain (orc2-cdk6A orc6-cdk4A MCM7-2NLS pGAL1-
), we observed three active S phase origins reinitiating by 2-D gel electrophoresis (Nguyen et al., 2001
). To comprehensively examine this prediction throughout the genome, we performed microarray CGH on the re-replicating DNA from OMC cells. This re-replicating DNA (flow cytometry in ) was competitively hybridized against DNA from a congenic non-re-replicating strain that lacks the inducible Δntcdc6 and will be referred to as the OM strain (orc2-cdk6A orc6-cdk4A MCM7-2NLS pGAL1
). Another source of non-re-replicating control DNA is OMC DNA from G1 phase cells, and when this was used, virtually identical results were obtained (unpublished data).
Figure 2. Re-replication induced during G2/M phase when ORC, Mcm2-7, and Cdc6 are deregulated. (A) G2/M phase re-replication in the OMC strain is readily detectable by flow cytometry. The OMC strain YJL3248 (orc2-cdk6A orc6-cdk4A MCM7-2NLS pGAL1-Δntcdc6 (more ...)
The OMC G2/M phase re-replication profiles are shown in and Supplementary Figure S7. These data confirm that the incomplete re-replication observed by flow cytometry is distributed over all 16 chromosomes, as was first suggested by their limited entry into the gel during PFGE (Nguyen et al., 2001
and ). The re-replication profiles also show that individual chromosomes re-replicate very unevenly, with some segments preferentially re-replicating more than others do.
Application of a peak finding algorithm to OMC re-replication profiles identified 106 reinitiating origins. Most of these origins appear to correspond to chromosomal loci that form pre-RCs in G1 phase because more than 80% of the reinitiating origins map to within 10 kb of a pro-ARS identified by Wyrick et al.
) as sites of pre-RC binding. The mean distance between the OMC reinitiating origins and the closest Wyrick pro-ARS (Wyrick et al., 2001
) is 7.0 kb. This value is highly significant (p < 5 × 10-8
) when compared with the mean distances calculated for equivalent numbers of randomly selected chromosomal loci, as the mean distances are tightly distributed around a value of 12.3 kb (Supplementary Figure S8).
Tanny et al.
) have analyzed the re-replication profile of a strain similar to our OMC strain containing the additional perturbation of a mutation of an RXL motif in ORC6 that abrogates CDK binding and results in a slightly increased extent of re-replication. Although both articles use slightly different data analysis and presentation, (our profiles are presented to preserve absolute copy number information at the cost of less distinctive peaks), the re-replication profiles are strikingly similar (compare Supplementary Figure S7 to Tanny et al., 2006
; Supplementary Figure S2). Like our results, 80% of the 123 re-replication origins identified by Tanny et al.
) are within 10 kb of a Wyrick et al.
) pro-ARS, further supporting the notion that re-replication occurs at normal sites of pre-RC formation. Overlap of origins identified in both studies is considerable, with 64% of the origins in this study within 10 kb of an origin in Tanny et al.
) (20% would be expected by chance). This overlap becomes even more striking, 80% overlap (expected value is also 20%), when the top 40 highest peaks in our analysis are compared with peaks identified in Tanny et al.
). Together with our previous confirmation by 2-D gel electrophoresis that ARS305, ARS121, and ARS607 reinitiate (Nguyen et al., 2001
), these genomic data suggest that reinitiation primarily occurs at a subset of potential S phase origins.
The efficiency with which these potential origins reinitiate in G2/M phase, however, does not correlate with the efficiency or timing with which they initiate in S phase. For example, only 38% of the active S phase origins reinitiate with enough efficiency to be identified as peaks during re-replication in G2/M phase. Moreover, some regions that normally replicate late in S phase, such as those near the telomeres of chromosome III, re-replicate very efficiently in G2/M phase, apparently from very inefficient or latent S phase origins in those regions. For a systematic comparison of re-replication efficiency versus replication timing of all potential S phase origins, we plotted the re-replication copy number versus the replication copy number for the set of pro-ARSs identified by Wyrick et al.
) (). The absence of any significant correlation (R2
= 0.0002) indicates that the efficiency or timing of a replication origin in S phase does not determine its re-replication efficiency during G2/M phase.
Mechanisms That Prevent Re-replication at G2/M Phase Also Act in S Phase
The prevailing model for replication control depicts the prevention of re-replication in S, G2, and M phase as one continuous inhibitory period using a common strategy of preventing pre-RC reassembly. Because CDKs are active throughout this period, the model would predict that mechanisms used by CDKs to regulate replication proteins should prevent re-replication throughout S, G2, and M phase. To determine if CDK regulation of ORC, Mcm2-7, and Cdc6, which prevents re-replication within G2/M phase, also prevents re-replication in S phase, we induced Δntcdc6 in OMC cells (orc2-cdk6A orc6-cdk4A MCM7-2NLS pGAL1-Δntcdc6) as they entered S phase.
OMC cells were arrested in G1 phase with α factor, and half the cells were harvested to obtain G1 phase DNA. The remaining cells were induced to express Δntcdc6 and then released from the G1 arrest into a low concentration of HU to delay their replication and allow us to collect them in S phase. Flow cytometry indicated that the released cells were harvested while still in S phase with a DNA content of 1.4 C (). The S phase and G1 phase DNA were competitively hybridized against the yeast genomic microarray to generate a combined replication/re-replication profile for S phase ( and Supplementary Figure S9).
Figure 3. Deregulation of ORC, Mcm2-7, and Cdc6 can induce re-replication in S phase. (A) Flow cytometry of OMC cells induced to re-replicate in S phase. The OMC strain YJL3249 (orc2-cdk6A orc6-cdk4A MCM7-2NLS pGAL1-Δntcdc6 pMET3-HA3-CDC20) was arrested (more ...)
Because normal S phase replication can account for an increase in DNA copy number from 1 to 2, only DNA synthesis beyond this copy number can be unequivocally attributed to re-replication. As seen in and Supplementary Figure S9, many early origins acquired a DNA copy number greater than 2; in some cases reaching values greater than 3. In the same profiles other chromosomal regions had copy numbers significantly below 2, confirming that cells were indeed in the midst of S phase. In fact, early origins reinitiated, whereas forks from their first round of replication were still progressing and before many late origins had fired. Similar re-replication profiles were observed for re-replicating cells synchronously harvested in S phase in the absence of HU (unpublished data). These findings thus directly establish that mechanisms used to prevent re-replication in G2/M phase also act within S phase.
Cell Cycle Position Can Affect the Extent and Location of Re-replication
To determine if the block to re-replication is modulated during progression through the cell cycle, we compared the re-replication profile of OMC cells (orc2-cdk6A orc6-cdk4A MCM7-2NLS pGAL1-Δntcdc6) that were induced to re-replicate through a complete S phase with the profile associated with re-replication in G2/M phase. To obtain the former profile, both OMC and control OM cells (orc2-cdk6A orc6-cdk4A MCM7-2NLS pGAL1) were arrested in G1 phase with α factor followed by addition of galactose to induce Δntcdc6 in the OMC strain. Cells were then released from the G1 arrest, allowed to proceed through S phase, and collected at a G2/M arrest 3 h after the release. DNA prepared from the OMC and OM strains were competitively hybridized to our yeast genomic microarray to obtain a “G1 release” re-replication profile for the OMC cells.
Flow cytometry showed that both the re-replicating OMC and the control OM strain were in the middle of S phase 1 h after the release (). As expected for actively replicating chromosomes (Hennessy and Botstein, 1991
), the chromosomes of these strains were retained in the wells during PFGE (). Two hours after the release, S phase was mostly complete in the control OM strain and its chromosomes reentered the gel during PFGE. In the OMC strain, however, the induction of re-replication prevented chromosomes from reentering the PFGE gel at both 2 and 3 h time points. Because significant re-replication could be induced in OMC cells delayed in S phase, we believe that re-replication during the progression through S phase contributed to the re-replication seen in the G1 release experiment.
Figure 4. Re-replication induced upon release from a G1 arrest when ORC, Mcm2-7, and Cdc6 are deregulated. (A) Robust re-replication of OMC cells after G1 release. The OMC strain YJL3248 (orc2-cdk6A orc6-cdk4A MCM7-2NLS pGAL1-Δntcdc6 pMET3-HA3-CDC20) and (more ...)
Re-replication induced during G1 release of OMC cells was more extensive than re-replication induced in G2/M phase. Despite comparable lengths of induction, flow cytometry reproducibly indicated that the former accumulated a DNA content of 3.2 C, whereas the latter accumulated only 2.7 C (compare 3 h time points in with ). More extensive re-replication could also be seen by comparing the re-replication profiles induced during the G1 release ( and Supplementary Figure S10) and the G2/M phase arrest ( and Supplementary Figure S7). In general the peaks in the G1 release profiles were taller than the G2/M phase profiles, suggesting that more efficient or more rounds of reinitiation can occur when re-replication is induced during S phase. For example, ARS305 reached a copy number of 6.6, indicating it reinitiated a second time, as a single round can only generate a maximum copy number of 4. Overall, multiple rounds of reinitiation were observed on more than half of the chromosomes when re-replication was induced during the G1 release. In contrast, multiple rounds of reinitiation occurred at much fewer loci and to a lesser extent when re-replication was induced in G2/M phase.
A peak finding algorithm identified 87 potential reinitiation sites when re-replication was induced during the G1 release experiment. Of these, 85% were located within 10 kb of a Wyrick pro-ARS Wyrick et al.
). These data suggest that re-replication induced during a G1 release occurs from S phase origins of DNA replication.
In addition to the extent of re-replication, another significant difference between re-replication induced during the G1 release and re-replication induced during G2/M phase was their pattern of origin usage. As discussed above, efficiency of re-replication in G2/M phase was not correlated with origin usage during S phase. In contrast, the efficiency of re-replication induced during the G1 release exhibited a modest positive correlation with S phase origin timing (). Although we cannot rule out an intrinsic difference in the reinitiation efficiency of early versus late origins when re-replication is induced during the G1 release, the simplest explanation for this correlation is that earlier replicating origins are cleared of pre-RCs earlier, making them available sooner for reassembly of pre-RCs and reinitiation within S phase.
Limited Re-replication Is Detectable with Fewer Genetic Perturbations
Our previous analysis of budding yeast re-replication failed to detect re-replication when only two pre-RC components were deregulated in G2/M phase (Nguyen et al., 2001
). This observation is frequently cited as evidence that eukaryotic replication controls are highly redundant. Both the increased sensitivity of the microarray CGH assay and the enhanced re-replication observed during a G1 release provided opportunities to reexamine whether these controls are indeed redundant in budding yeast.
As a first step, we examined an “OC” strain (orc2-cdk6A orc6-cdk4A pGAL1-
), in which only ORC and Cdc6 are deregulated and compared it with a control “O” strain (orc2-cdk6A orc6-cdk4A GAL1
), where only ORC is deregulated. In accordance with our previous results (Nguyen et al., 2001
), induction of Δntcdc6 in G2/M phase generated no significant increase in DNA content by flow cytometry () or chromosome immobilization during PFGE (). Similarly, microarray CGH of DNA prepared from the OC and O strains after 3 h of galactose induction in G2/M phase detected no re-replication on 15 out of 16 chromosomes (Supplementary Figure S11). However, limited re-replication could clearly be observed on both arms of chromosome III (). Thus, the microarray CGH assay can detect re-replication missed by other assays.
Figure 5. Re-replication can be induced when only ORC and Cdc6 are deregulated. (A) Re-replication is undetectable by flow cytometry in OC cells in G2/M phase. The OC strain YJL3240 (orc2-cdk6A orc6-cdk4A pGAL1-Δntcdc6 pMET3-HA3-CDC20) and the control O (more ...)
We next asked whether we could detect more re-replication in the OC strain by inducing it during a G1 release. In contrast to the results obtained during a G2/M phase induction, significant re-replication was detected by flow cytometry and PFGE within 2 h of the G1 release (). The re-replication profile of the OC strain induced during a G1 release ( and Supplementary Figure S11) showed broad re-replication zones of ~200-500 kb in width on all chromosomes. These results, along with the re-replication induced during G2/M phase, establish that deregulating just ORC and Cdc6 is sufficient to induce re-replication and thus these inhibitory mechanisms are not truly redundant. The greater amount of re-replication induced during G1 release versus G2/M arrest underscores the dynamic character of the block to re-replication and, in this case, is likely due to the incomplete expulsion of Mcm proteins from the nucleus during S phase.
Microarray CGH Can Detect Re-replication Initiating Primarily from a Single Origin
To further investigate the question of redundancy in replication control, we examined the consequences of deregulating just Mcm2-7 and Cdc6. We were not able to detect re-replication in the “MC” strain (MCM7-2NLS pGAL1-
) whether Δntcdc6 was induced in G2/M phase or during a G1 release (unpublished data). Hence, we further deregulated Cdc6 inhibition by mutating the two full CDK consensus phosphorylation sites on Δntcdc6 to generate the MC2A
). These additional mutations increase the stability of Δntcdc6 (Perkins et al., 2001
Expression of Δntcdc6-cdk2A in the MC2A strain in either G2/M phase or during a G1 release did not cause a detectable increase in DNA content by flow cytometry (). However, PFGE suggested that chromosome III re-replicated in a small subset of MC2A cells when Δntcdc6-cdk2A was induced under either protocol (). Microarray CGH provided definitive evidence that re-replication occurred, in this strain, primarily on the right arm of chromosome III ( and Supplementary Figure S12).
Figure 6. Re-replication occurs primarily on a single chromosome when Mcm2-7 and Cdc6 are deregulated. (A) Re-replication is undetectable by flow cytometry in MC2A cells in G2/M phase. The MC2A strain YJL4489 (MCM7-NLS pGAL1-Δntcdc6-cdk2A pMET3-HA3-CDC20 (more ...)
To confirm that the very limited DNA re-replication in the MC2A strain arose from a canonical reinitiation event, we asked whether this re-replication depended on known origins and initiation proteins. Our peak finding algorithm implicated an initiation event at ~297 kb, close to ARS317, an inefficient S phase origin located at 291 kb. Two-dimensional gel analysis of ARS317 () detected bubble arcs, indicative of replication initiation, in the MC2A strain but not the control “M” strain (MCM7-2NLS pGAL1). The immediately adjacent origins, ARS316 and ARS318, only displayed fork arcs (unpublished data), suggesting that most of the re-replication on the right arm of chromosome III originates from ARS317. Deletion of ARS317, but not ARS316 or ARS318, in the MC2A strain eliminated the bulk of the re-replication detected by microarray CGH ( and unpublished data), demonstrating that re-replication initiates primarily from a single S phase origin.
Figure 7. The re-replication arising from deregulation of both Mcm2-7 and Cdc6 depends on ARS317 and Cdc7. (A) Reinitiation bubbles are induced at ARS317 when MC2A re-replicates in G2/M phase. The MC2A strain YJL4489 (MCM7-NLS pGAL1-Δntcdc6-cdk2A pMET3-HA3-CDC20 (more ...)
We next asked whether this re-replication is dependent on the essential initiation factor, Cdc7-Dbf4 kinase. Both MC2A and MC2A cdc7-1 strains were induced to re-replicate in G2/M phase under permissive (23°C) and restrictive (35°C) temperatures for the cdc7-1 allele. Microarray CGH demonstrated that both strains re-replicated to a similar extent at 23°C (Supplementary Figure S13), but at 35°C there was little or no re-replication in the MC2A cdc7-1 strain (). Together, the dependence on both ARS317 and Cdc7-Dbf4 indicates that the very limited re-replication induced in the MC2A strain arises primarily from a single bona fide reinitiation event.