The first step in the initiation of DNA replication is the binding to origins of the heterohexameric Origin Recognition Complex (ORC). Subsequently ORC, in collaboration with other proteins, promotes the binding of another heterohexameric complex composed of MiniChromosome Maintenance (MCM) proteins. The combination of ORC, the MCM complex, and certain additional proteins is called a "pre-replication complex" (pre-RC). During S phase, initiation of replication is triggered by Cyclin-Dependent Kinase (CDK) and Dbf4- (or Dfp1-) Dependent Kinase (DDK) at origin-bound pre-RCs (reviewed in ).
In the budding yeast, Saccharomyces cerevisiae, replication origins consist of 100–200 bp of DNA, which include an 11-bp AT-rich ORC-binding consensus sequence (reviewed in ). In other studied eukaryotic organisms, origins are not so clearly defined. For example, S. pombe origins are larger (500–2000 bp) and have no apparent consensus sequence. However an AT-rich region is necessary for ORC binding [2–4], and asymmetric AT-rich sequence motifs (with A residues primarily in one strand and T residues primarily in the complementary strand) are present and redundantly contribute to origin function [5–10]. In contrast to the high efficiency of many S. cerevisiae origins, S. pombe origins display a wide range of efficiencies, and few, if any, are capable of firing in more than 70% of S phases [11–15]. It appears that replication origins in other eukaryotic organisms share with S. pombe origins the characteristics of absence of a consensus sequence and frequent inefficiency (reviewed in [16, 17]). That is, a large number of sequences have the potential to function as replication origins, but only a small subset of such sequences are used by a single cell in any given cell cycle, and the subset selected is highly variable from cell to cell.
Previously, a small number of S. pombe replication origins was studied intensely by autonomously replicating sequence (ARS) assays, which identify the cis-acting DNA sequences important for origin function when the origin region is relocated to a plasmid. A few S. pombe replication origins were also studied by two-dimensional (2D) agarose gel electrophoretic analyses, which permit measurements of replication fork direction and firing efficiency of origins in their endogenous chromosomal locations. These investigations revealed that most of these previously studied S. pombe origins fire early in S phase. However, a few sequences with positive activity in ARS assays proved to be replicated in late S phase – primarily passively – by forks coming from nearby earlier-firing origins . The studied late-replicating potential origins include ars727, ars2-2, and the telomere-associated sequences at the heterochromatic ends of chromosomes 1 and 2 (; A. Chaudari and J. A. Huberman, unpublished). Surprisingly, most of the heterochromatic regions of the S. pombe genome – centromeres, the silent mating type locus, and ribosomal DNA (rDNA) – proved to replicate in early S phase. The only tested heterochromatic region that replicates in late S phase is the telomeres [18, 19].
The conclusions listed in the preceding three paragraphs were based on studies of about 20 origins. To find out whether these conclusions applied to all S. pombe origins or just a subset, two laboratories have recently used genome-wide computer analyses. Analyzing the few known S. pombe origins led Segurado et al.  to conclude that origins have an unusually high A+T content. They developed an algorithm based on AT content to generate a list of 387 predicted origins (which they called "AT islands"), each of which contains an unusually high A+T content distributed over a broad region (up to 1 kb). Eighteen of twenty predicted origins that were randomly selected from their list of 387 had detectable in vivo initiation activity when tested by 2D gel electrophoresis, suggesting that this simple computational approach could be a surprisingly accurate predictor of origin locations in S. pombe. Dai et al.  noticed that the base composition and sequence properties of known origins (high AT; frequent runs of A or T residues) closely resembled those of the longer and more AT-rich intergenic regions. Indeed, when they tested all of the intergenic regions in a 68-kb stretch on chromosome 2, they found that 14 of the 26 intergenic regions exhibited detectable ARS activity. The remaining inactive intergenic regions were shorter and/or less AT-rich than the active ones. These results suggested that about half of S. pombe intergenic regions (about 2500) may occasionally function as origins. This number is much greater than the previous estimates of 250  to 700 [22, 23] origins in the genome. The large number of potential origins in the genome, combined with the fact that 10 of the 14 active ARS elements in the 68-kb studied region displayed only weak activity suggested that origin usage in single cells during single S phases may be determined stochastically .
Indeed, the conclusion that replication origins in S. pombe are generally inefficient and fire stochastically, without relationship to the firing frequency of their neighbors and without relationship to their firing in previous generations, was strengthened by the results of another genome-wide approach, DNA fiber fluorography, which directly demonstrated the low efficiencies of individual origins and the lack of coordination between them (i.e., stochastic firing) .
Other laboratories have employed microarrays to obtain genome-wide information about the locations and efficiencies of origins in synchronized fission yeast cells entering S phase in the presence of HU, which starves cells for dNTPs and thus slows replication fork movement. Under these conditions, the replication checkpoint is activated. For this reason, all of the microarray analyses have compared results from wild-type cells with results from cells bearing mutations that inactivate the replication checkpoint.
The replication checkpoint responds to stalled replication forks (for example, forks that stall when cells are treated with HU or with a DNA-alkylating agent such as methyl methane sulfonate, MMS). Stalled forks, in combination with other proteins, activate an upstream kinase, Rad3 in S. pombe or Mec1 in S. cerevisiae. Both are homologues of mammalian ATR. The upstream kinase activates a downstream kinase, Cds1 in S. pombe or Rad53 in S. cerevisiae. These are structural homologues of mammalian Chk2 and functional analogs of both Chk2 and Chk1. Indeed, when replication forks stall as a consequence of template DNA methylation by MMS, the replication checkpoint is activated, and progress through S phase is slowed in both budding [24, 25] and fission yeast [26–29]. Impeding replication forks also leads to checkpoint-dependent replication slowing in mammalian cells [30–32].
In mammalian cells, checkpoint-dependent slowing of S phase is accomplished by a combination of checkpoint-dependent inhibition of origin firing [16, 31–33] and checkpoint-dependent inhibition of replication fork movement [32, 33]. However, in budding yeast the slowing of S phase in response to MMS treatment is accomplished entirely by inhibition of replication origin firing; no checkpoint-dependent fork inhibition is detectable . The mechanism by which S phase is slowed in MMS-treated fission yeast cells is not clear. The evidence available before the advent of microarray studies suggested that, as in budding yeast, slowing of replication in fission yeast probably depended on inhibition of origin firing, because, when the replication checkpoint is activated by treating S. pombe cells with HU, the replication of two late potential origins (ars2-2 and telomeres) is retarded in checkpoint-dependent fashion . However, results from the new microarray studies, which are reviewed below, have not been entirely consistent with each other or with these prior studies and have led to greater confusion.
In the first microarray study, Feng et al.  took advantage of the fact that replication forks unwind parental strands, generating single-stranded DNA (ssDNA). Thus, if cells enter S phase in the presence of HU, those origins capable of firing in early S phase generate small regions of single-stranded DNA (ssDNA), while regions far from early-firing origins remain double-stranded. Feng et al.  looked for regions of ssDNA in both HU-treated S. cerevisiae and in HU-treated S. pombe cells. Furthermore, they examined both wild-type cells and replication-checkpoint-mutant cells.
To study the effects of the replication checkpoint on origin firing in S. cerevisiae, Feng et al.  compared HU-treated wild-type with HU-treated rad53 mutant cells. They found that 2/3 of S. cerevisiae replication origins are restrained from firing by the replication checkpoint. An even more recent study, which employed copy number measurements rather than measurements of single-stranded DNA, confirmed that 2/3 or more of S. cerevisiae replication origins are checkpoint-restrained .
When Feng et al.  applied the same procedure to S. pombe and smoothed the results over a 12-kb window, 321 ssDNA peaks (putative origins) were identified in cells lacking the Rad53 homologue, Cds1. Of these, 125 (39%) were specific to cds1 Δ cells (apparently checkpoint-restrained). These observations suggested, therefore, that in S. pombe a smaller (but still significant) proportion of origins is restrained by the replication checkpoint than in S. cerevisiae. In both yeasts, ssDNA accumulation was much greater in the checkpoint-deficient strains, presumably due to the roles of Cds1 and Rad53 in stabilizing replication forks (reviewed in ).
In the second microarray study, Heichinger et al.  measured changes in copy number at each position in the genome as a synchronized population of fission yeast cells entered S phase in the absence or presence of HU. The investigators made similar measurements for checkpoint-mutant (rad3 Δ) cells in the presence of HU. The results of all three experiments were in good agreement with each other, and they permitted the identification of 401 relatively strong origins plus 503 putative weaker origins. Surprisingly, only about 2% of these origins appeared to be inhibited by the replication checkpoint – in contrast to the ssDNA measurements of Feng et al. , which had suggested that ~39% of fission yeast replication origins are checkpoint-restrained.
The third microarray investigation, by Hayashi et al. , provided additional important information but no clear resolution to the apparent conflict between the checkpoint-mutant results of Feng et al.  and Heichinger et al. . Hayashi et al.  used immunoprecipitation in combination with microarrays to localize pre-RCs (binding sites for both ORC and MCM polypeptides). A total of 460 pre-RCs was found. Then microarrays were used to identify those pre-RCs where significant incorporation of the thymidine analogue, 5-bromo deoxyuridine (BrdU), took place when synchronized cells entered S phase in the presence of HU. Of the 460 pre-RCs, 307 incorporated significant BrdU and were considered to be early-firing and/or strong origins. Little or no BrdU incorporation was detected at the remaining 153 pre-RCs, which were consequently characterized as late-firing and/or weak origins. Hayashi et al.  found that there was increased incorporation of BrdU at about 22% of origins (mostly weak/late origins) in checkpoint-mutant (cds1 Δ) cells compared to wild-type cells, and such checkpoint-mutant-induced BrdU incorporation was especially frequent in sub-telomeric regions. However, at most of these apparently checkpoint-inhibited origins, the extent of BrdU incorporation in cds1 Δ cells was significantly less than for the early/strong class of origins and could only be detected by dividing the signal from cds1 Δ cells (small) by the signal from wild-type cells (even smaller) .
Thus the three microarray studies appeared to reach rather different conclusions regarding the extent to which fission yeast replication origins are restrained by the replication checkpoint. The two studies that appeared to detect a large fraction (22% or 39%) of checkpoint-restrained origins were both based on comparisons between wild-type and cds1 Δ cells. The single study that found only a small fraction (2%) of checkpoint-restrained origins employed rad3 Δ cells. Each of the three studies employed a different procedure to measure extents of replication in wild-type and checkpoint-mutant cells. Could these experimental differences explain the different results obtained?
Here we present the results and conclusions of our own microarray-based measurements of DNA replication, as synchronized wild type, cds1 Δ and rad3 Δ cells enter S phase in the presence of HU. Our results provide an explanation for the differences between the earlier microarray studies. Our findings also lead to interesting conclusions regarding the control of replication timing, the mechanisms by which a subset of origins is restrained from firing in HU-treated cells, the mechanism by which fission yeast cells retard S phase in response to DNA damage, and the relationships between checkpoint regulation of origins and their chromatin structure.