earlier in S phase are enriched in the pool compared to sites that replicate later (Figure 2A) and the relative abundance of a given site in the S phase pool is inversely proportional to its average relative time of replication during S phase [9]. The results were visualized by plotting the relative enrichment in the S phase sample for all array features along a given chromosome, termed a replication timing profile (Figure 2B and Figure S2, red line). Peaks in the profile result from regions that replicated before neighboring sequences, and therefore must contain pre-meiotic origins of replication. We next sought to compare the subset of origins that are active during meiS and mitS. We created pre-mitotic replication profiles for cells of the same genetic background (SK1) using alpha-factor synchronized cultures (Figure 2B and Figure S2, blue line). The mitS replication profiles were very similar to those obtained in W303, another S. cerevisiae strain background (Figure S3), indicating that our method works similarly to those previously published and that the replication program is robust across different strains. To measure the level of random noise inherent in the copy number technique, we performed a control co- hybridization of two G1 DNA samples (Figure 2B and Figure S2, grey line). Because the smoothing algorithm used to create the profiles is unable to accurately predict replication time at the end of chromosomes, we excluded the 25 kb at each chromosome end from our analyses. After this exclusion, the meiS and mitS replication profiles showed a high degree of similarity (Pearson correlation coefficient = 0.64). Importantly, the peaks in both profiles were associated with sites of G1 Mcm2-7 binding (Figure 2B, inverted triangles), indicating that we could identify active origins of replication in both profiles. In general, the same peaks were present in both the pre-meiotic and pre-mitotic replication profiles, extending to all chromosomes the previous observations that the same origins initiate DNA replication during both meiS and mitS [13,14]. Although we observed several instances in which meiosis- or mitosis-specific origins initiated replication (Figure S2), the effect on the overall profile was minimal, suggesting that these differences do not contribute substantially to S-phase progression. Replication initiation is delayed for many pre-meiotic origins Although the majority of origins were active in meiS and mitS, examination of the replication profiles revealed that the relative replication time was different at many sites. The observation that many of the peak heights differed suggested that the timing or efficiency of replication initiation is altered at some origins in the meiotic cell cycle. When the distribution of replication timing for potential origins (Mcm2-7 binding sites) was compared to the whole genome in mitS, the majority of origins were replicated before bulk genomic DNA (Figure 2C, compare blue and black lines on right distribution). This finding is consistent with a model of efficient DNA replication initiation from these sites as cells enter mitS. Replication of potential origins was very limited at the end of S phase, consistent with this replication being comprised of fork progression and termination. In contrast, potential origin replica- tion distributed more uniformly throughout meiS (Figure 2D, compare red and black lines), indicating that all origins did not initiate replication as efficiently upon entry into meiS. To further investigate the delay in replication initiation in meiS, we compared the relative time of replication of all Mcm2-7 sites in mitS and meiS (Figure 2D). Given that there is no specific marker for the start and end of S phase in individual cells, or even the population as a whole, we used the FACS analysis to estimate the minimum length of meiS was 45 minutes at 30uC (Figure 2A), Separating DNA Synthesis and Meiotic DSB Formation approximately twice as long as mitS [24]. If origins initiated replication at a similar time during meiS and mitS, we would expect the distribution of origin timing to resemble the orange dashed line (Figure 2D). If the time of replication were scaled with the length of S phase, but origins still fired in essentially the same order, we would expect the data to cluster around the blue linear- scaling prediction line (Figure 2D). We found that origins fired in a similar order in meiS and mitS, but 71% of potential origins replicated relatively later in meiS than mitS (Figure 2D, dots above the blue line). Consistently, we observed that 74% of origins replicated before the mean of the genome (mid-S phase) in mitS, but this number was reduced to 58% in meiS. We created a best fit model using linear regression (Figure 2D, solid purple line) [25], and found that the earlier origins were most delayed and the replication rate seemed to increase at the end of S phase, similar to predictions from other eukaryotes [26]. These data indicate that replication of many potential origins is delayed in meiS, either because of later initiation or passive replication in late S phase. Pre-meiotic cells have compromised replication capacity To confirm the apparent differences in replication kinetics that we observed in the meiS and mitS replication profiles, we sought to identify all early replicating origins by delaying cells in early meiS with hydroxyurea (HU), as previously described for mitotic cells [9]. To begin, we tested a titration of HU concentrations to determine the optimal conditions to use with sporulating cells. We found that pre-meiotic cells were more sensitive to replication inhibition than mitotically dividing cells. First, the nature of the replication arrests differed. Pre-meiotic cells treated with 20– 200 mM HU exhibited little or no replication progression, indicating that they had largely arrested replication in very early S phase (Figure 3A, SPO samples). In contrast, the mitotic cells inoculated into rich medium (YPD) containing 5–200 mM HU did not fully arrest DNA replication. Consistent with previous reports [27], even in the highest concentration of HU we observed detectible DNA replication after 2 hours (Figure 3A, YPD+200 mM HU). To further test the idea that cells are more sensitive to HU exposure in meiS, we measured the autophos- phorylation associated with activation of the intra-S phase checkpoint kinase Rad53 by western blotting. Consistent with the idea that pre-meiotic cells are more sensitive to replication inhibition, Rad53 became hyper-phosphorylated at a much lower concentration of HU in meiS than mitS (Figure 3B). Because it has been reported that cells sporulated in the presence of HU have reduced levels of early meiotic transcripts, including IME2 [28], we were concerned that addition of HU may generally inhibit meiotic entry independent of DNA replication and S-phase checkpoint activation. To test whether cells had entered meiS in each of our experiments, we measured the CDK- dependent phosphorylation of Orc6 that occurs as cells enter S phase. We were able to detect Orc6 phosphorylation consistent with meiS entry in cells exposed to 5–200 mM HU, although the kinetics of Orc6 phosphorylation were delayed in the presence of high amounts of HU (Figure 3C). Addition of 200 mM urea, a similarly nitrogen-rich compound, also slowed meiS DNA replication and S-phase entry (Figure 3A, 3C). However, because this treatment did not inhibit mitS DNA replication (Figure 3A) or elicit a checkpoint response in meiS or mitS (Figure 3B), we conclude that high concentrations of HU or urea inhibit meiotic cell cycle entry independent of S-phase checkpoint activation. When HU- or urea-treated cells were released into sporulation medium without HU, they completed meiosis and formed viable spores, indicating they were reversibly inhibited and remained viable during the treatment (data not shown). Together, these data PLoS Genetics | www.plosgenetics.org 4 May 2012 | Volume 8 | Issue 5 | e1002643