Re-replication Initiates at Distinct Sites in the Genome
To assess the extent of re-replication across the S. cerevisiae
genome, we used DNA microarrays to determine changes in DNA copy number as cells underwent re-replication. This technique has been used previously to identify sites of replication initiation by detecting newly synthesized DNA as cells pass through S-phase (Raghuraman et al., 2001
; Yabuki et al., 2002
). Our experiments were conducted using an S. cerevisiae
strain with mutations that overcome all currently known mechanisms that prevent reinitiation (Wilmes et al., 2004
). Re-replication in this strain is controlled using a galactose-inducible, nondegradable Cdc6. To ensure that all observed replication was due to re-replication, cells were arrested in G2/M before the induction of re-replication (). DNA was isolated from re-replicating cells at various time points after addition of galactose (). Unreplicated DNA isolated from G1-arrested cells served as a hybridization reference. The two populations of DNA were differentially labeled and cohybridized to a high-density DNA microarray with 44,000 features distributed throughout the genome (Pokholok et al., 2005
). Initial experiments showed that cells after 3 h of Cdc6 induction had the most significant re-replication (), thus this time point was used in all subsequent experiments.
Figure 1. Multiple pre-RC mutations result in induced re-replication. (A) An outline of the re-replication experiment. Re-replication-sensitive cells were grown to an OD600 of 0.4 in YPD and then arrested in nocodazole. After the cells were arrested, galactose (more ...)
Analysis of three independent experiments showed that re-replication occurs at specific sites in the genome. To visualize the sites of re-replication, the log ratio of re-replicated/unreplicated DNA for each spot on the array was plotted as a function of its position along the chromosome (). The resulting profiles have distinct peaks, identifying sequences present in elevated copy number and that have re-replicated. Control experiments using strains lacking the genetic changes required for re-replication showed no significant variation in DNA copy number across the genome (Supplementary Figure 1).
Figure 2. Analysis of genome-wide re-replication. (A) Re-replication is detected by copy number analysis using DNA microarrays. DNA from re-replicating cells and from G1-arrested cells was differentially labeled and cohybridized to a high-density DNA microarray. (more ...)
Re-replication Initiates from Sites of G1 pre-RC Formation
Peaks in the re-replication profile represent the most frequently re-replicated sequences, suggesting that they are sites of reinitiation. To determine if these sites are coincident with previously identified, potential origins, we compared the re-replication profile to sites of G1 pre-RC formation in wild-type cells as determined by genome-wide location analysis of Mcm2-7. This comparison allowed us to determine if peaks of re-replication colocalized with sites that have the capability to initiate replication during S-phase.
To compare the re-replication profile and G1 Mcm2-7 binding sites, we determined the midpoint of the peaks in each of the data sets. Before analysis, we applied a smoothing algorithm to the re-replication profile to help delineate the peaks by reducing random noise (, gray histogram, Supplementary Figure 2). Initial analysis showed that peaks on the re-replication profile substantially overlapped sites of Mcm2-7 binding (, black histogram, Supplementary Figure 3). To conduct a more quantitative analysis, a peak-finding algorithm was used to define the mid-points of the peaks along the chromosome in both data sets. We monitored the overlap between peaks on the re-replication profile and sites of Mcm2-7 binding using a range of window sizes and found that a 7.5-kb window was optimal. Using this window size, 82% of re-replication peaks overlapped with Mcm2-7 binding sites (). We noted that the peak-finding algorithm was not able to identify all sites of re-replication and thus possible reinitiation (e.g., peaks at chromosome ends; see Supplementary Figure 4). Accounting for these uncalled initiation sites in the re-replication data set, the final percent of re-replication peaks that are within 7.5 kb of an Mcm2-7 binding site is 91%. We conclude that re-replication largely occurred at sites that normally direct pre-RC formation during G1.
Comparison of overlap of peaks between all data sets
Although nearly all sites of re-replication overlapped with Mcm2-7 binding sites, the converse was not true. There were many sites of G1 pre-RC formation that did not align with peaks of re-replication. Using the same computational-based analysis as described above, we found that only 31% of all sites of pre-RC formation during G1 showed significant re-replication (). Together, our data show on a genome-wide level that not all potential sites of initiation can re-replicate and, therefore, that the extent of protection from re-replication is not uniform across the genome.
Origins Direct Re-replication
To directly address if origin sequences are required for re-replication, we asked if moving an origin sequence associated with a peak of re-replication was sufficient to establish a new site of re-replication in the genome. These experiments focused on the ARS418
locus, which is a site of G1 pre-RC formation (, black histogram), provides origin function on a plasmid (unpublished data), is a peak on an S-phase timing curve (Raghuraman et al., 2001
; Yabuki et al., 2002
; MacAlpine and Bell, 2005
and unpublished data), and is closely associated with a prominent peak on the re-replication profile (, gray histogram). Six hundred base pairs surrounding the ARS418
locus were integrated at an ectopic intergenic region (iYDR309C
) that showed little, if any, re-replication (, gray histogram and see , closed circles). Using the re-replication-sensitive strain containing the ectopic ARS418
, we performed the same re-replication experiment described above. The resulting re-replication profile showed that the insertion of ARS418
at the iYDR309C
locus induced substantial re-replication (, gray histogram) compared with the strain without the ectopic ARS418
(, dashed line). We also ectopically inserted 200 base pairs surrounding another re-replicating origin into iYDR309C
(see dotted black line on Chromosome II in Supplementary Figure 2 for location). This second origin also induced re-replication at iYDR309C
(Supplementary Figure 5). Thus, moving only origin-proximal DNA is sufficient to direct re-replication at a new locus.
Figure 3. An origin sequence directs reinitiation (A) The re-replication profile surrounding iYDR309C, a segment that does not re-replicate, in the absence of an ectopic origin is depicted as both the gray histogram and the black dashed line. (B) ARS418, an origin (more ...)
Figure 5. Origins are capable of reinitiating multiple times. (A) Diagram of density transfer experiment. A cartoon depicts what products will look like during the experiment with “heavy” DNA strands shown in black and “light” DNA (more ...)
To demonstrate that the origin was necessary for the new re-replication peak, we mutated the ectopically inserted ARS418
so that it was no longer functional. Our laboratory recently refined an algorithm (Breier et al., 2004
) to identify functional ARS Consensus Sequences (ACS) across the S. cerevisiae
genome (our unpublished results). Using this algorithm, we predicted the site of the essential ACS of ARS418
and mutated this sequence. This mutation eliminated the function of ARS418
on a plasmid (unpublished data). The mutant ARS418
was integrated at iYDR309C
and the re-replication of this strain was analyzed by microarray (, gray histogram). Unlike the wild-type ARS418
, the mutant ARS418
did not induce re-replication at iYDR309C
, showing that the same sequence that is required for origin function in a plasmid context during normal S-phase is also required to direct re-replication. This observation is consistent with previously published data concerning ARS305
(Nguyen et al., 2001
Timing of Initiation during S-phase Does Not Correlate with the Ability to Re-replicate
Having demonstrated that sites of reinitiation correspond to a subset of potential origins, we asked if sites of reinitiation represented a particular class of origins. We compared origins that reinitiate to the time of initiation of those same origins during S-phase. We used a previously described protocol (Yabuki et al., 2002
) to identify origins that initiated in the presence of HU (Supplementary Figure 3). HU allows early origins to initiate but inhibits activation from later-initiating origins of replication (Santocanale and Diffley, 1998
; Shirahige et al., 1998
Comparing the profile generated in the presence of HU with the re-replication profile showed that some of the reinitiating origins are early, but not all. Similarly, there are early origins that do not re-replicate. Using a window of 7.5 kb and computationally based analysis, 48% of re-replication peaks are associated with HU-initiating origins (). Conversely, 52% of HU-initiating origins re-replicate. These data suggest that there is not a strong correlation between origins that re-replicate and when that origin initiates during S-phase. Thus, the factors that determine timing of initiation in S-phase are not the same as the factors that sensitize origins to re-replication during G2/M.
The telomeres and centromeres of the S. cerevisiae genome are specialized regions of the genome that replicate at specific times during S-phase (telomeres replicate late, whereas centromeres replicate early), so we also analyzed the ability of these regions to re-replicate. The subtelomeric chromosomal regions appeared overrepresented in the re-replicated fraction of the DNA (Supplementary Figure 2). To examine this feature further, we plotted the relative level of re-replication for each point on the array as a function of its distance from the telomere (, black plot). For comparison, we plotted the relative level of re-replication for a wild-type strain under re-replicating conditions (, gray plot). The resulting plot showed a positive correlation between the proximity of a sequence to the telomere and its extent of re-replication. We also plotted each point on the array as a function of its distance from the centromere (, black plot) and found that there was no correlation between distance from the centromere and sensitivity to re-replication.
Figure 4. Subtelomeric regions have a high probability of re-replicating. (A) There is a positive correlation between the proximity of a sequence to the telomere and its probability of re-replicating. The relative enrichment for each spot on the microarray was (more ...)
Origins Can Reinitiate Multiple Times
FACS analysis 3 h after induction of re-replication shows that most cells in the population have ~3C DNA content; however, some cells appear to have DNA content greater than 4C (). The existence of cells with >4C DNA content suggests that at least a subset of origins is capable of multiple rounds of reinitiation. To determine if origins can reinitiate more than once, we used a density transfer approach to more accurately determine the extent of re-replication at particular regions.
Cells were labeled with dense isotopes as outlined in . As illustrated, at the nocodazole arrest, cells will have passed through S-phase and therefore have one heavy and one light DNA strand. Induction of re-replication in the nocodazole-arrested cells will result in a third species of DNA composed of entirely light DNA strands. If a segment of DNA re-replicates exactly once then the ratio of Light-Light (LL) DNA to Heavy-Light (HL) DNA will be 1:1. If a segment has re-replicated more than once, the ratio will increase.
We examined several sites that represented different features of the re-replication profile to determine their extent of re-replication. We tested two origins that were prominent sites of reinitiation (ARS418 and ARS428; see ), two origins that did not seem to be efficient sites of reinitiation (ARS1 and ARS1413; see and Supplementary Figure 2), and one sequence that was not substantially re-replicated, iYDR309C (). Consistent with their prominence in the re-replication profiles, both ARS418 and ARS428 have at least twice as much LL DNA as HL DNA (, closed triangles and open squares). Consistent with the re-replication profile, these data definitively demonstrate that some origins are capable of reinitiating multiple times. iYDR309C, however, showed no LL DNA, indicating that other regions of the genome do not re-replicate at all. Together, these data strongly support the model that re-replication is limited across the genome but that origins that reinitiate can do so more than once.
Pre-RC Formation Is Not the Only Determinant of the Ability to Re-replicate
We have shown that not all sites of G1 pre-RC formation reinitiate. Because previous data strongly suggest that Mcm2-7 loading onto origin DNA is required for reinitiation (Nguyen et al., 2001
), there are two possible explanations for only a subset of these G1 pre-RC sites undergoing re-replication. First, it is possible that Mcm2-7 is only recruited to those origins that reinitiate. Alternatively, similar to the pre-RCs assembled in G1, Mcm2-7 could load at all potential origins, but only a subset is competent to reinitiate. To distinguish between these hypotheses, we asked where pre-RCs were formed during re-replication using Mcm2-7 genome-wide location analysis. To avoid confusing sites of pre-RC formation with fork movement, samples were taken 45 min after induction when re-replication is limited as determined by FACS () and array analysis (unpublished data).
We first asked if Mcm2-7 binds to the same sites during re-replication as seen during G1. Because both genome-wide location analysis data sets have narrow peaks (compared with the re-replication profile), we could use a much smaller window when comparing G1 and re-replication Mcm2-7 binding sites. Using a 1-kb window, 92% of the re-replication Mcm2-7 binding sites overlap with G1 Mcm2-7 binding sites (). In contrast, only 45% of G1 Mcm2-7 binding sites overlap with re-replication Mcm2-7 binding sites (, , and Supplementary Figure 3), demonstrating that only a subset of sites that assemble pre-RCs in G1 also do so in re-replicating cells.
Figure 6. Recruitment of Mcm2-7 is not sufficient for reinitiation. (A) Mcm2-7 binds only a fraction of possible origins during re-replication. Genome-wide location analysis of Mcm2-7 and ORC was performed 45 min after induction of re-replication. The binding sites (more ...)
We were concerned that Mcm2-7 associated with a subset of origins during re-replication because in the re-replication-sensitive strain, which has several ORC mutations, ORC only associated with the same subset of origins. To determine the location of ORC binding during re-replication, we performed ORC genome-wide location analysis as described above ( and Supplementary Figure 3). We found that the majority of G1 Mcm2-7 binding sites overlap with sites of re-replication ORC binding sites (), suggesting that ORC containing two nonphosphorylatable subunits can bind to most potential origins. Therefore, ORC binding does not limit Mcm2-7 loading. Similar to G1 Mcm2-7 binding sites, only 52% of re-replication ORC binding sites are associated with a re-replication Mcm2-7 binding site. Thus the reduction in pre-RC formation during re-replication is not due to a reduced number of ORC binding sites.
We then determined how many sites of reinitiation overlap with re-replication Mcm2-7 binding sites. We used the same approach to compare these two data sets as when we compared the re-replication profile to G1 Mcm2-7 binding sites. We found that 71% of the re-replication profile peaks overlapped with a re-replication Mcm2-7 peak within a 7.5-kb window (). Taking into account the peaks that were not identified by the peak-finding algorithm (Supplementary Figure 4), the percentage increased to 80%. These comparisons show that Mcm2-7 is found at most sites of reinitiation, supporting the model that Mcm2-7 is required at origins that reinitiate.
We then asked what percentage of re-replication Mcm2-7 binding sites overlapped with sites of re-replication. Fiftyone percent of re-replication Mcm2-7 binding sites overlapped with re-replication peaks (), suggesting that only a subset of sites that exhibit Mcm2-7 association during induced re-replication go on to reinitiate ( and Supplementary Figure 3). We also measured the interorigin distance between sites of re-replication as well as the distance between pre-RC binding during re-replication (Supplementary Figure 5). The median distance between origins that initiate during re-replication is 84 kb, but the median distance between Mcm2-7 binding sites during re-replication is only 57 kb. Thus, there are substantially more Mcm2-7 binding sites during induced re-replication than there are re-replication initiation sites.
These data support the first hypothesis presented above, which stated that reinitiation was limited to sites that load Mcm2-7 during re-replication. We found, however, that loading of Mcm2-7 was not sufficient to induce reinitiation because there were many origins throughout the genome that loaded Mcm2-7 but did not re-replicate (). With respect to the ability to re-replicate, sites of G1 pre-RC formation can be grouped into three classes: those that do not form pre-RCs during re-replication, those that form pre-RCs but do not reinitiate, and those that form pre-RCs and reinitiate. The recruitment of Mcm2-7, therefore, is not the only obstacle to re-replication and there must be other levels of control that act after pre-RC formation to prevent reinitiation.