replicating or G1-arrested cells were labeled. Samples were labeled with Cy3-dUTP and Cy5-dUTP by random priming using 4 mg random nonamer oligo (IDT) and 10 units of Klenow (New England Biolabs, Beverly, MA). Unincorporated dye was removed using microcon columns (30-kDa MW cutoff, Millipore, Bedford, MA), and samples were co-hybridized to custom Agilent arrays (Wilmington, DE) using a standard protocol. Microarray data analysis For each co-hybridization, Cy3 and Cy5 levels were calculated using Agilent Feature Extractor CGH software. Background normalization, log2 ratios for each experiment and scale normalizations across each set of triplicate experiments were calculated using the sma package [62] in R, a computer language and environment for statistical computing (v2.1.0, http://www.r- project.org). The raw data and log ratios analyzed in this study are available from the NCBI Gene Expression Omnibus (http://www. ncbi.nlm.nih.gov/geo/), accession number GSE35667. Mcm2-7 binding sites were defined as sites that were significantly enriched (P,0.05) in 3 independent experiments. Array features within 500 bp of each other on the chromosome were merged into a single binding site. Mcm2-7 binding sites were assigned to a previously characterized origin if they overlapped the defined region (http://www.oridb.org). Clear Mcm2-7 peaks were detected at some sites that did not make the statistical cutoff. They were manually included in the list of binding sites if they corresponded to a known origin and an Mcm2-7 binding site was called in the other data set (meiotic or mitotic). For analysis of replication timing, Mcm2-7 sites were defined as the ACS, when one was predicted [63] or previously defined (http://www.oridb. org), or else as the midpoint of the minimally defined origin region. The replication time of each Mcm2-7 binding site was determined for each experiment by assigning it to the time of the closest point on the smoothed and predicted replication timing curve. Points ,25 kb from chromosome ends were excluded from timing analysis due to the inability to accurately predict timing at chromosome ends. For pooled S-phase and HU DNA samples, DNA replication profiles were smoothed and predicted every 50 bp using the loess smoothing spline with a span = 0.025 and a spar = 0.45 (Table S5). Mcm2-7 binding sites were considered replicated in HU if their value was greater than half of the maximum value in the genome (excluding points ,25 kb from chromosome ends). Axis association sites were defined as those with overlapping Rec8, Hop1 and Red1 binding at more that one adjacent chromosomal feature, defined similarly to Mcm2-7 binding sites, except with P,0.15. The positions of axis association sites were taken to be the midpoint of the intersection of the binding peaks of all three proteins. Analysis of the replication time for axis association sites, Spo11 binding sites and ssDNA-enriched sites was performed as for the Mcm2-7 binding sites. Gene expression analysis To measure gene expression, we analyzed the average of expression values at 15 and 20 minutes post alpha-factor from the mitotic data set of Granovskaia at al. [30] and the average of the 2- and 3-hour expression data for wild-type cells from Borde et al. [31]. The expression data sets were scale-normalized to a mean log value of 0 and a standard deviation of 1 over the 4987 genes for which data were available in both sets. Origin-proximal transcripts are those within 500 base pairs of the Mcm2-7 binding site. We repeated all analyses using the dataset of Primig et al. [64] and Separating DNA Synthesis and Meiotic DSB Formation Friedlander, et al. [65], and obtained highly similar results (data not shown). Indirect immunofluorescence on spread nuclei Meiotic nuclear spreads were performed as described [66]. In brief, the nuclei of spheroplasted cells were spread on a glass slide in the presence of paraformaldehyde fixative and 1% lipsol. After drying, the slides were blocked in blocking buffer (0.2% gelatin, 0.5% BSA in PBS) and stained with anti-Red1 (N. Hollingsworth, 1:250 dilution). Analysis of ssDNA enrichment The genome-wide analysis of DSBs in the cdc6-mn strain using ssDNA enrichment was conducted as described [36]. CHEF and Southern analysis Clamped-homogeneous electric field (CHEF) gel electrophoresis and Southern blotting were performed as described [36]. Supporting Information Figure S1 Mcm2-7 localization. Mcm2-7 enrichment was plotted versus chromosome position for all 16 chromosomes for meiotic cells (red, enrichment is upwards) and mitotic cells (blue, enrichment is downwards). Inverted triangles represent the significant binding sites we identified. White dots indicate the position of the centromere on each chromosome. (TIF) Figure S2 MeiS and mitS replication profiles. The smoothed, predicted replication timing profiles are shown for each of the 16 chromosomes for meiS (red lines), mitS (blue lines) and G1 vs. G1 control hybridization (grey lines). Mcm2-7 binding sites are indicated by inverted triangles for meiotic (red) and mitotic (blue) cells. Black dots indicate the positions of centromeres. (TIF) Figure S3 Comparison of mitS replication profiles for 2 yeast strains. MitS replication profiles for all chromosomes are shown for the current study in SK1 (blue lines) and for W303 (green lines) from Yabuki and colleagues [9]. Black dots indicate the positions of centromeres. (TIF) Figure S4 HU replication profiles. The extent of replication after 4 hours in HU is plotted for all 16 chromosomes for wild- type cells (H574) in YPD+200 mM HU (blue lines), SPO+20 mM HU for wild-type cells (H574, red lines), sml1D cells (H4898, purple lines), rec8D (H5187, orange lines) and spo11D (H5184, green lines). Inverted arrowheads denote origins that are replicated in each strain of the corresponding color. Black dots indicate the positions of centromeres. (TIF) Figure S5 Comparison of timing and HU profiles. The extent of replication after 4 hours in HU is plotted for all 16 chromosomes for wild-type cells (H574) in YPD+200 mM HU (blue histogram) and SPO+20 mM HU for wild-type cells (H574, red histogram). Above each histogram the corresponding S phase replication timing profile is plotted in blue for mitS and red for meiS. Black dots indicate the positions of centromeres. (TIF) Figure S6 DSB hotspot replication time. (A) The distribution of relative replication time for HSs mapped by Spo11-oligo recovery are shown for all 3434 non-telomeric HSs (grey box), and the 3434 HSs ranked in quintiles from lowest break number (purple) to PLoS Genetics | www.plosgenetics.org 11 May 2012 | Volume 8 | Issue 5 | e1002643