Separating DNA Synthesis and Meiotic DSB Formation Figure 3. Reduced replication initiation in meiS. Pre-sporulation cultures of wild-type cells (H574) were inoculated into either SPO (top row) or YPD (bottom row) in the absence or presence of the indicated concentrations of HU or urea. (A) Comparison of response to HU in meiS and mitS as measured by FACS analysis. (B) Western blot analysis of Rad53 phosphorylation after 4 hours incubation in SPO (top panel) or YPD (bottom panel). (C) Western blot analysis of Orc6 phosphorylation in cells from the SPO cultures to monitor activation of CDK at the time of S phase entry. (D) The relative copy number enrichment of cells after 4 hours in 200 mM HU (mitS) or 20 mM HU (meiS) is plotted relative to chromosome VII position for wild-type (H574, blue for mitS and red for meiS) and sml1D cells (H4898, purple). (E) The total number of origins replicated in each of the conditions in (D) is represented as a Venn diagram. doi:10.1371/journal.pgen.1002643.g003 indicate that high concentrations of HU (or urea) can reversibly delay meiS entry. Therefore, we chose to use 20 mM HU for all further meiotic experiments, as this concentration of HU inhibited meiS progression without significant delays in meiotic cell cycle entry. We used HU to determine the number and location of early- replicating origins in meiS and mitS. To create as similar a situation as possible, we synchronized cells in G1 in pre- sporulation medium, and subsequently divided the cultures into either sporulation medium (to induce meiosis) containing 20 mM HU or rich medium (to induce mitosis) containing 200 mM HU. After four hours, total DNA was collected and relative copy number was measured genome-wide. We detected replication initiation at a subset of sites in both pre-meiotic and pre-mitotic HU-arrested cells (Figure 3D, Figure S4 and Figure S5). The extent of replication in HU for each origin was similar to the time of replication of that site in the corresponding S-phase replication profile. The highest peaks in the HU profiles almost always coincided with the highest peaks in the corresponding replication timing curve (Figure S5). Thus, both the S phase and HU profiles detected the locations of early replicating origins. We compared the identity of origins replicated in meiotic and mitotic cells exposed to HU. We considered all origins that showed copy number enrichment greater than half the maximum enrichment of the genome to be replicated in each HU experiment (Figure 3D and Figure S5, inverted triangles). Although the majority of these origins were associated with a clear peak in the HU profiles, indicative of active initiation, some of these origins also could have been passively replicated by the fork from a nearby origin. Consistent with previous results from mitotically dividing cells, we observed that all chromosomes contained multiple early- replicating origins in mitS. Most chromosomes contained early- replicating origins during meiS, although the sites on chromo- somes VIII and XVI were just below the 50% cutoff in the meiS HU profile. Comparing the number of origins replicated in the meiS and mitS HU profiles revealed that many fewer origins initiated replication in HU in pre-meiotic cells (47 versus 121, Figure 3E and Table S1). All origins that were replicated in HU in pre-meiotic cells were also replicated in HU in pre-mitotic cells (Figure 3E), indicating that a subset of early mitotic origins also function efficiently in meiS, but that others become inhibited by HU during the sporulation program. PLoS Genetics | www.plosgenetics.org 5 May 2012 | Volume 8 | Issue 5 | e1002643