levels of DSBs form in cdc6-mn cells, which complete very little DNA replication (Figure 6), extending our previous finding that cdc6-mn cells form DSBs at the engineered his4-LEU2 hotspot [54]. It has been observed that Spo11 is first loaded onto centromeres in meiS, before it becomes redistributed to sites along chromosome arms [19], and it is possible that the early replication of centromeres drives Spo11 association. However, we were unable to detect the specific replication of centromeres in cdc6-mn cells by CGH analysis (data not shown), yet they were able to form full levels of DSBs, indicating that Spo11 loading does not require early centromere replication. Although the wild-type kinetics of DSB formation in cdc6-mn cells indicates that chromosome axis assembly and DSB formation proceed on a timer that does not require DNA replication, it is clear that locally delaying DNA replication by eliminating origins on one chromosome arm retards proximal DSB formation [20,55]. The data presented here indicate that DSB formation is not intrinsically dependent on replication fork passage. The apparent discrepancy of these results could be explained if the single, severely delayed replication fork on the origin-less chromosome arm were to locally disrupt the loading or phosphorylation of DSB factors, delaying the initiation of DSB formation. In this case, the depletion of replication forks in the cdc6-mn cells would allow DSB formation. Alternatively, the processes could be coupled through a checkpoint mechanism triggered by the single delayed replication fork. In mitotic cells, the intra-S phase checkpoint down-regulates the activity of DDK [56,57]. As it was demonstrated that DSB formation requires higher levels of DDK activity than DNA replication [47], the intra-S phase checkpoint could prevent DSB formation in wild- type meiotic cells, but may be lacking in cdc6-mn cells due to the severe decrease in DNA replication, similar to results obtained in S. pombe [58,59]. In either model, DNA replication is not a prerequisite for DSB formation, but rather the processes would be coordinated by superimposed regulatory mechanisms. Whereas much effort has been made to understand the events of meiotic prophase, relatively little is known about the regulation of meiS and the assembly of specialized chromosome structures necessary for meiotic recombination. In many organisms, the signals that initiate meiosis are unclear, and meiotic cell cycle entry cannot be determined by molecular or cytological markers until after the initiation of recombination. Studies in S. pombe revealed that pre-meiotic DNA replication initiates from the same sites as mitotic DNA replication, but replication is delayed or less efficient for a significant number of origins [15]. Our results indicate that this delay is due to limiting replication capacity, and reveal that centromeres are a strong determinant of early replication timing. Additionally, in S. pombe DNA replication is also not required for DSB formation, as multiple mutants that decrease DNA replication form DSBs across all chromosomes [58]. Therefore, the modular regulation of DNA replication and meiotic chromo- some formation is conserved across distantly related yeast species, and could extend to other organisms as well. Materials and Methods Strains and growth conditions Strains used in this study are isogenic to SK1 and are listed in Table S4. Gene disruptions were carried out using a PCR-based protocol [60]. FLO8 was deleted in SB1505 to reduce flocculation. Cells lacking CLN3 were used for Mcm2-7 Genome-wide location analysis experiments to increase efficiency of meiotic entry. To induce synchronous meiosis, strains were pre-inoculated at OD600 = 0.2 in YPA (1% yeast extract, 2% bactopeptone, 1% Separating DNA Synthesis and Meiotic DSB Formation potassium acetate, Figure 1 and Figure 2), or BYTA medium (1% yeast extract, 2% tryptone, 1% potassium acetate, 50 mM potassium phthalate, Figure 3, Figure 4, Figure 5, Figure 6), grown for 16 hours at 30uC, washed twice with water, and resuspended at OD600 = 1.9 in SPO medium (0.3% potassium acetate, pH 7.0) at 30uC. For G1 control DNA, mitotic Mcm2-7 chromatin immunoprecipitation and mitS phase profiles, cells were inoculated into fresh YPD medium containing 5 mg/ml alpha-factor for 3 hours. For mitS profiles, cells were released into S phase by washing with 2 volumes of sterile water and resuspended in YPD at 30uC. MeiS cells containing the ime2-as1 allele were synchronized by incubation in SPO+20 mM 1-NA-PP1 (Toronto Research Chemicals) for 4 hours, the cells were washed with 2 volumes of sterile water and resuspended in SPO medium. Samples of 1.5 mls were removed every 5 minutes for mitS profiles and every 7.5 minutes for meiS profiles. All DNA samples inclusive of S phase (those which showed cells in S phase in the FACS profiles, as well as two time points before and after) were pooled for processing. For hydroxyurea (HU) experiments, cells were inoculated into YPD containing 200 mM HU or SPO containing 20 mM HU for 4 hours at 30uC. Chromatin immunoprecipitation 25 mls of cells were harvested after 1 hour (Mcm2-7) or 3 hours (Rec8, Hop1, Red1) in SPO. For pre-mitotic Mcm2-7 analysis, 50 mls of culture at OD600 = 0.8 were collected from alpha-factor arrested cultures. Chromatin immunoprecipitation (ChIP) was performed as described [61]. One tenth of the lysate was removed as an input sample. Samples were immunoprecipitated for 16 hours at 4 degrees with UM185 (Rabbit polyclonal anti- Mcm2-7, 2 ml serum used per immunoprecipitation), 3F10 (Rec8- 3HA, Roche, used 2 mg per immunoprecipitation), anti-Hop1 or anti-Red1 (N. Hollingsworth, 2 ml each serum used per immuno- precipitation). FACS analysis Cell pellets from 100 ml of sporulation culture were fixed in 70% ethanol overnight at 4uC. Ethanol was removed and cells were resuspended in 500 ml of 50 mM sodium citrate (pH 7.0) containing 20 mg RNaseA for 16 hours at 50uC. Subsequently, 100 mg of Proteinase K were added and samples were incubated an additional 24 hours. 500 ml of 50 mM sodium citrate (pH 7.0) containing 2 mM Sytox green (Invitrogen) were added. Cells were sonicated briefly on lowest power and scanned using a FACSCa- libur (BD Biosciences). DNA extractions for CGH Cells were lysed by bead beating in 500 ml phenol/chloroform and 500 ml of breakage buffer (10 mM TRIS, pH 8.0, 1 mM EDTA, 100 mM NaCl, 2% Triton X-100, 1% SDS). After centrifugation, the aqueous phase was precipitated with ethanol and resuspended in 500 ml TE7.6 (10 mM TRIS, pH 7.6, 1 mM EDTA) with 30 mg RNase. DNA was resuspended and samples were incubated at 50uC for 3 hours. DNA was sheared by sonicating at 100% output and lowest power for 10 seconds using a Branson sonicator. DNA was extracted with 500 ml phenol/ chloroform, precipitated with ethanol and resuspended in 100 ml of TE7.6 Fluorescent labeling and microarray hybridization For ChIP experiments, one half of the immunoprecipitated DNA and one tenth of the input DNA were labeled. For pooled S-phase and HU replication profiles, ,5 mg of DNA from PLoS Genetics | www.plosgenetics.org 10 May 2012 | Volume 8 | Issue 5 | e1002643