Author Summary Sexually reproducing organisms rely on a specialized cell division called meiosis to produce genetically distinct gametes with half the chromosome number of the parent. The first stage of the meiotic cell division is the duplication of chromosomes, followed by the exchange of DNA between homologous chromosomes inherited from both parents. It has long been known that DNA replication occurs more slowly in pre-meiotic cells than in mitotically dividing cells, and it was postulated that this delay is due to the chromosome structures or proteins required for homolo- gous DNA exchange. We show here that the delay of DNA replication in yeast is regulated separately from the formation of recombinant chromosomes; preventing re- combination structures from forming does not alleviate the delays in pre-meiotic DNA replication, and cells lacking DNA replication are able to initiate recombination. We propose that these two processes are functionally separable and that the delay in pre-meiotic DNA replication in yeast may be a result of the starvation conditions required for the induction of meiosis in this organism. suggested that origin selection is also similar in both S phases in fission yeast [15]. In budding yeast there is no clear separation of meiS and the start of prophase; DNA synthesis occurs concurrently with the loading of factors required for axis and DSB formation, and both require the same cell-cycle kinase activities. The meiosis-specific cohesin complex containing Rec8 is loaded onto chromosomes as cells enter meiS, and subsequently the axial proteins Hop1 and Red1 associate with the same axial core sites along each chromosome [16,17]. As cells progress into prophase, chromo- somes condense into a characteristic form, with a shortened axis and intervening DNA loops emanating away from the central core (reviewed in [2]). Association of both axial and DSB factors with core sites is critical for the formation of DSBs on the adjacent loops by the topoisomerase-like enzyme Spo11 [17,18], whose proper association is dependent on Rec8 [16,19]. A possible link between axis morphogenesis and S phase length was inferred from FACS analysis of total DNA content in yeast strains lacking Rec8 and Spo11 [12], and conversely, DNA replication timing has been implicated as a determinant of the time of DSB formation [20]. To better understand how the early meiotic cell division is coordinated, we characterized the kinetics and requirements of meiS and axis formation genome-wide in budding yeast. We found that origin firing was either delayed or less efficient at the majority of origins in meiS. Consistent with a decreased replication capacity, cells were more sensitive to nucleotide depletion during meiS. However, preventing meiotic chromosome reorganization had little effect on origin activation in meiS, suggesting that DNA replication is not strongly regulated by or linked to axis structure. Conversely, full DNA replication was not required for either axis assembly or DSB formation. Together, these data indicate that DNA replication and the initiation of homologous recombination are separable events, which coordinately contribute to the formation of meiotic recombinant chromosomes. Results Differential Mcm2-7 loading at a subset of pre-meiotic origins To determine whether the initial selection of potential replication origins could explain the difference in S phase length between the Separating DNA Synthesis and Meiotic DSB Formation meiS and mitS, we performed genome-wide location analysis for the Mcm2-7 helicase in pre-meiotic and pre-mitotic cells (Figure 1A and Figure S1). In total from both experiments, we observed Mcm2-7 binding at 393 loci, of which 382 had been identified previously as Figure 1. Transcription influences origin selection. (A) Mcm2-7 localization was performed for cln3D cells (A4224) prior to meiosis and a wild-type strain (SB1505) prior to mitosis. Mcm2-7 enrichment was plotted versus chromosome position for chromosome IX for meiotic cells (red, enrichment is upwards) and mitotic cells (blue, enrichment is downwards). Inverted red and blue triangles indicate significant Mcm2- 7 binding sites. Black arrowheads indicate the positions of ARS913 and the SPO22 ARS. (B) As in (A), except a detailed view of ARS913 (left) and the SPO22 ARS (right) as indicated by arrowheads. The schematic above indicates the locations of coding regions. (C) Quantification of the change in gene expression for genes next to meiosis-specific (red), mitosis-specific (blue) and all other (white) Mcm2-7 binding sites. (D) Histogram showing the distribution of the calculated distances between Mcm2-7 binding sites prior to meiosis (red, left panel) and mitosis (blue, right panel). doi:10.1371/journal.pgen.1002643.g001 PLoS Genetics | www.plosgenetics.org 2 May 2012 | Volume 8 | Issue 5 | e1002643