Figure 2. Meiotic DNA replication profiles. (A) Ime2-as1-myc homozygous diploid cells (KBY518) were synchronized in meiS. DNA samples were collected every 7.5 minutes. Resulting samples were pooled and co-hybridized with a G1 DNA sample to a tiled genomic microarray. (B) Replication profiles for meiS (KBY518, red line), mitS (SB1505, blue line) and control G1 vs. G1 (SB1505, grey line) hybridizations were created by plotting the smoothed log2 ratio (see Materials and Methods) versus chromosome VII position. Triangles indicate the positions of Mcm2-7 binding sites prior to meiosis (red) and mitosis (blue). (C) The distribution of relative replication time for all origins (colored lines) and for the entire genome (black lines) is plotted for meiS (left panel) and mitS phase (right panel). (D) The replication time in meiS of Mcm2-7 binding sites that were present in both cell cycles were plotted as a function of mitS replication time. Assuming meiS is twice as long as mitS, the orange dashed line indicates the predicted meiS replication time if origins replicated with the same Separating DNA Synthesis and Meiotic DSB Formation kinetics in meiS and mitS. The blue dashed line is the predicted replication time trend line if scaling were linear with respect to S phase length. The purple solid line is the second order polynomial best-fit model. doi:10.1371/journal.pgen.1002643.g002 potential replication origins in mitotic cells in multiple strain backgrounds (Table S1). Comparison of the pre-meiotic and pre- mitotic Mcm2-7 binding sites revealed that the majority of origins loaded Mcm2-7 in both pre-meiotic and pre-mitotic cells (358/393, Table S1), consistent with the hypothesis that the mechanism of origin selection is the same in both cell cycles. Although origin selection was conserved at most sites, we observed differential Mcm2-7 binding at 35 sites; 22 mitosis-specific and 13 meiosis-specific sites (Table S1 and Table S2). Sites where Mcm2-7 binding differed between pre-meiotic and pre-mitotic cells were more frequently located in coding regions or promoters than sites with similar Mcm2-7 binding in both cell cycles (20/35 versus 87/358, Chi-squared p = 8.1E-5). Origins are generally under- enriched in coding regions of the genome due to the incompatibility between transcription and replication factor binding [21]. Consis- tent with this incompatibility, mitosis-specific Mcm2-7 binding sites were found in sporulation-induced genes SPO22 and ZIP1, and meiosis-specific binding sites were associated with mitotic budding- related genes SHE2 and BUD27 (Figure 1B and Table S2). Moreover, we observed significantly increased gene expression during meiS at mitosis-specific Mcm2-7 binding sites, compared to sites that bind Mcm2-7 in both cell cycles (Figure 1C compare blue and white boxes, t-test p = 1.7E-2), suggesting that differential Mcm2-7 binding at many of these sites was driven by changes in gene expression. Meiosis-specific Mcm2-7 binding sites did not show as clear a change in gene expression, suggesting that other mechanisms may also contribute to Mcm2-7 association. Never- theless, the small number of changes in Mcm2-7 binding we observed are unlikely to account for the extended length of meiS, as we did not observe larger gaps between Mcm2-7 binding sites in meiotic cells (Figure 1D). Indeed, the meiosis- and mitosis-specific Mcm2-7 binding sites were consistently located close to other origins; the next Mcm2-7 binding site was on average 14 kb away with a maximum distance of 39 kb. In comparison, the average and maximum inter-origin distance for all potential origins was 30 kb and 95 kb, respectively (Figure 1D). These data indicate that the reduced rate of meiS is not due to differential origin selection. The same origins are active in the meiotic and mitotic cell divisions Another possible explanation for the extended timing of meiS is that fewer sites with loaded Mcm2-7 complexes are used as origins of replication in meiS. To determine which potential replication origins were ‘‘active’’ during meiS, we measured the average replication time of sites across the genome in sporulating cells. To synchronize the cells as they passed through meiS, we used an ATP-analog-sensitive allele of the Ime2 kinase, ime2-as1 [22]. Ime2 promotes meiotic cell cycle entry and pre-meiotic DNA replication [22,23], so ime2-as1 cells inoculated into sporulation medium containing the ATP-analog inhibitor for 4 hours did not initiate DNA replication (Figure 2A, 0 minutes). When the inhibitor was removed, cells progressed synchronously through meiS, as measured by FACS (Figure 2A). To determine the relative time of DNA replication, we pooled DNA samples that were collected every 7.5 minutes from the start to the end of meiS. The resulting samples were applied to a microarray together with a control (non- replicating) G1 sample to determine the relative abundance of DNA at 40,646 sites across the genome. Because the quantity of the DNA doubles when a site is replicated, sites that replicate PLoS Genetics | www.plosgenetics.org 3 May 2012 | Volume 8 | Issue 5 | e1002643