Given that meiotic cells were far more sensitive to inhibition of DNA replication by HU treatment (Figure 3A), we asked whether low nucleotide levels could explain the delayed replication initiation in meiS. We increased nucleotide levels by removing the ribonucleotide reductase (RNR) inhibitor SML1 [29]. When we measured DNA replication in sml1D cells treated with HU, we found that increasing nucleotide levels increased the number of early origins to levels intermediate between meiS and mitS conditions (total of 71, Figure 3E and Figure S4). SML1 deletion did not result in defects in meiotic S phase entry, sporulation efficiency or spore viability (data not shown). Given the sensitivity of meiotic cells to HU treatment, and the increases in DNA replication observed when nucleotide levels are increased, we propose that the starvation conditions required to initiate meiotic entry lead to low intra-cellular nucleotide levels that delay DNA replication. Centromeres are a strong determinant of early replication in meiS In an attempt to explain the changes in replication initiation timing that occurred between meiS and mitS, we looked at the relationship between chromosomal features and replication timing. Because meiotic entry is associated with large changes in the gene expression program, we first explored the connection between replication timing and gene expression. Using published datasets, we determined the expression of all genes within 500 bp of meiS and mitS origins [30,31]. We found no relationship between meiotic gene expression level and replication time in meiS (Figure 4A). We also examined expression of genes surrounding the 47 meiS early origins and the 74 mitS-only early origins that do not initiate replication in HU in meiS. We found there was no significant difference between the expression levels of genes adjacent to these two classes of origins (Figure 4B, compare red and blue boxes), again suggesting that the delay in meiS replication initiation is not due to transcriptional changes proximal to these origins. Similarly, we tested for a correlation between changes in time of replication and changes in gene expression between meiotic and mitotic cells, but found no relationship (data not shown), indicating that the large-scale changes in replication timing in meiS are independent of the meiotic gene expression program. Finally, we explored the locations of meiotic unanno- tated transcripts (MUTs) [32] and found no relationship between their presence and Mcm2-7 binding and origin activation (Table S2). For example, ECM23/MUT1498 is predicted to cover ARS1621, yet we observed Mcm2-7 binding and a peak in the replication profiles indicating origin activation at this site in both meiS and mitS (Figure S2). We next asked whether early replication of centromeres was conserved in meiS, because centromere proximal regions of chromosomes are replicated early during mitS in multiple yeasts [33,34]. Indeed, we found that all centromeres replicated in the first half of S phase in both mitS and meiS, with an average replication time of 22% and 28% of S phase, respectively (Figure 4C). Additionally, centromere-proximal origins were highly enriched in the meiS HU profiles for every chromosome (Figure S4). Plotting the replication time of all origins during meiS as a function of distance from the centromere revealed that origins close to centromeres were consistently replicated earlier in S phase than origins farther from centromeres. Strikingly, meiS early origins were on average 70 kb from a centromere, and the majority (32 of 47) was within 50 kb of a centromere (Figure 4D red dots, Figure 4E red distribution). Conversely, the set of origins that are replicated early during mitS extended significantly further (average of 176 kb) from centromeres (Figure 4D, blue dots, Separating DNA Synthesis and Meiotic DSB Formation Figure 4. Centromeres replicate early in S phase. (A) The average expression level of origin proximal genes is plotted versus the time of replication in meiS. The red dotted lines indicate the population average. (B) The expression level distributions for meiS (left) and mitS (right) are plotted for the genes surrounding each origin for meiS early origins (red boxes) and mitS-only early origins (blue boxes). (C) The replication time for each centromere is indicated as a gray vertical bar compared to the distribution of replication time for the whole genome (black line) in meiS (left panel) and mitS (right panel). The mean replication time of the genome is indicated by the black dotted lines for each panel. (D) The replication time of each origin is plotted as a function of the distance of the origin from the closest centromere. MeiS early origins are indicated in red, mitS-only early origins are indicated in blue and late origins are colored black. (E) The data from (D) are summarized as box and whisker plots, with significance of the difference between mei-S and mitS-only early origins indicated. doi:10.1371/journal.pgen.1002643.g004 Figure 4E, blue distribution, t-test p = 1.2E-4). The effect of centromere proximity on replication time extended 50–100 kb along the chromosomes, as the origins in this range replicated significantly earlier than those farther away (Figure 4C, 4D). The overall size of this 100–200 kb domain on each chromosome could, at least in part, explain why the smallest chromosome have a relatively high density of early origins and, on average, replicate PLoS Genetics | www.plosgenetics.org 6 May 2012 | Volume 8 | Issue 5 | e1002643