early (Table S1 and Figure S2). These data reveal that, as in mitS, centromeres are a strong determinant of early meiS replication initiation, and the effect is more apparent during meiS due to compromised DNA replication capacity. MeiS timing does not correlate with meiotic chromosome structure Given that meiotic chromosomes undergo large structural changes in preparation for recombination, and factors involved in these processes have been implicated in the control of meiotic replication, we investigated the relationship between pre-meiotic DNA replication and DSB formation. To understand whether axis or DSB formation delay meiS replication initiation, we measured early DNA replication (in the presence of HU) in cells unable to form meiotic axes (rec8D) or defective in DSB formation (spo11D). We observed similar HU replication profiles in sporulating wild- type, rec8D and spo11D cells: the vast majority of early meiS origins were replicated in all three strains (44 of 47 in wild-type cells, Figure 5A, 5B and Figure S4). These data indicate that Rec8 and Spo11 are not primarily responsible for the changes in meiotic replication origin timing that we observed. We next determined the replication time of several chromo- somal features during meiS, including DSB hotspots (HSs) and axis-associated core regions. Analysis of 3434 HSs mapped by Spo11-oligo accumulation [35] revealed that DSB sites were replicated throughout S phase (Figure 5C, grey line). When the HSs were ordered by rank, there was a slight trend that the stronger HSs were replicated earlier in S phase than the weaker sites, although the difference was not statistically significant (Figure S6A). We also measured the replication time of the strong DSB HSs mapped by either ssDNA enrichment in dmc1D cells or Spo11 genome-wide location analysis in rad50S cells [36] and found both were replicated with timing mirroring the entire genome (Figure 5C, brown and green lines, respectively, t-test p = 0.25 for dmc1D and p = 0.90 for Spo11 DSBs), indicating neither set of HSs are preferentially enriched in early or late replicating regions. Since many DSB factors associate with axis sites [17], we also measured the replication time of these regions. We defined axis association sites by overlapping localization of the axial proteins Rec8, Hop1 and Red1, which occurred at 565 sites in the genome (Figure S7, Table S3). As with HSs, axial sites were replicated throughout S phase, with a distribution similar to the whole genome (Figure 5D, t-test p = 0.97). Moreover, the change in timing of DSB and axis sites showed no trend toward earlier or later DNA replication (Figure S6B). The lack of detectable relationships between replication timing and the presence of axis and DSB sites suggests that meiotic chromosome structures do not strongly influence the timing of meiotic replication. DNA replication is not required for axial element association or DSB formation Since axis formation was not a critical determinant of meiS replication timing, we wondered whether replication timing might instead contribute to axis formation. Therefore, we monitored axis formation by indirect immunofluorescence of the Hop1 and Red1 proteins on spread nuclei from cells lacking complete DNA replication. We inhibited DNA replication in 3 ways; by arresting cells in early S phase with HU, by depleting the Mcm2-7 loading factor Cdc6 (cdc6-mn), which severely decreases DNA replication, and by removing the cyclins Clb5 and Clb6, which prevents all pre-meiotic DNA replication [37]. In each case we observed Hop1 and Red1 distributed along chromosomes (Figure 6A for Red1, Hop1 data not shown), demonstrating that replication is not Separating DNA Synthesis and Meiotic DSB Formation Figure 5. No relationship between DNA replication timing and recombination sites. (A) Chromosome VII replication profiles for pre- meiotic cells in the presence of 20 mM HU are shown for wild-type (H574, red), rec8D (H5187, orange) and spo11D (H5184, green) cells. Inverted triangles indicate the position of origins that are considered replicated in each strain. (B) Venn diagram summary of the experiment shown in (A), with the same color coding. (C) The distributions of replication timing in meiS are shown for the entire genome (black line), DSBs hotspots mapped by Spo11-oligo recovery (gray line), ssDNA enrichment in a dmc1D strain (brown line) and Spo11 binding in rad50S cells (green line). (D) The distributions of replication timing in meiS are shown for the entire genome (black line) and for axis association sites (blue line). doi:10.1371/journal.pgen.1002643.g005 required for meiotic axis association. We noted that the chromosomes failed to condense and individualize in the clb5D clb6D nuclei, indicating that CDK activity and/or DNA replication are likely important for the full assembly of normal meiotic chromosome structures. However, previous analysis of Rec8 staining in cdc6-mn cells indicated that full DNA replication is not required to form full axes [38,39]. To confirm that axis formation occurs on the same sites in the presence and absence of DNA replication, we localized Rec8, Hop1 and Red1 by whole- genome location analysis. As previously described, Hop1 (Figure 6B and Figure S7) and Red1 (Figure S7) localized to cohesin-associated regions (CARs) [17,40], similar to both Scc1 in PLoS Genetics | www.plosgenetics.org 7 May 2012 | Volume 8 | Issue 5 | e1002643