A. Formation and processing of meiotic DSBs.
B. Amplification of adaptor-ligated Spo11 oligos and mock immunoprecipitation processed in parallel.
C. Reproducibility of Spo11 oligo maps. Oligo counts are in hits per million mapped reads (hpM).
D. Proportions of reads mapped to unique regions and repetitive elements.
E. Quantitative agreement of Spo11 oligos and DSBs. Oligo counts in 19 hotspots were compared with DSBs (mean ± sd, 3 cultures) assayed by Southern blot of DNA from dmc1 and sae2 mutants.
F. The Spo11 oligo map agrees with dmc1 ssDNA microarray analysis (Buhler et al., 2007), but has higher resolution.
G. Agreement of the Spo11 oligo map with direct DSB detection in sae2 genomic DNA (spo11yf=DSB-deficient mutant spo11-Y135F. P=parental band, asterisk=cross-hybridization). Oligo counts in C,F,G were smoothed with a 201 bp sliding Hann window.

A. Hotspots near CYS3. Green bars are hotspots from this study. Hotspots from ssDNA mapping are also indicated (red and teal circles, peaks only (Buhler et al. 2007; Borde et al. 2009); orange circle and line, peak and region with signal over threshold (Blitzblau et al. 2007)).
B. Verification of an unusually broad hotspot by Southern blot of genomic DNA (labels as in Figure 1G). DSB signal in dmc1 spreads out because of hyperresection.
C. Hotspot intensities vary over a smooth continuum. Inset: cumulative fraction of hotspots vs. cumulative fraction of Spo11 oligos. Red lines mark the fraction of hotspot oligos in the weakest 67% of hotspots.

A, B. Mean normalized Spo11 oligo profiles (see Supplemental Experimental Procedures), smoothed with 75 bp window, and nucleosome occupancy (3 hr sample) around TF sites.
C. TFs clustered according to local spatial pattern of Spo11 oligos. Each horizontal line on the heat map shows the mean profile for binding sites of a single TF, grouped by k-means clustering. Because locally normalized oligo counts were used, color coding reflects DSB spatial pattern, not total DSB intensity.
D. Zones of protection from Spo11 or DNase I (Hesselberth et al. 2009) around Reb1 binding sites (n=156).
E. TFs that are most or least associated with frequent DSB formation nearby. TFs were rank ordered by mean oligo count ±500 bp from their binding sites. A subset of TFs is shown, others are in Figure S6B. Bars depict the fraction of sites not in hotspots (black) and divide the remainder according to hotspot quartile.

A. Models for origin of long and short oligos. i. Nicks (red arrows) positioned asymmetrically relative to Spo11. ii. Two DSB classes with symmetric nicks. iii. Variable nick placement, independent on left and right.
B. Asymmetry at DSB sites in the YCR048w hotspot (Chr III, 211, 744-211, 764 bp). Arrows show two known DSB sites that cannot be resolved due to ambiguity of oligos with 5′-C. Gray brackets, regions pooled to tally oligos for each strand.
C. Substantial asymmetry at Spo11 cleavage sites genome wide. Strand-specific counts were tallied for sites with ≥8 oligos total (n=49, 115). Points in red are significantly asymmetric (Poisson test, p≤0.05 after correction for false discovery). Green lines, 95% confidence intervals for Poisson sampling with equal frequencies on Watson and Crick.
D. Net asymmetry in the CYS3 hotspot.
E. Net asymmetry of hotspots genome wide (n=3, 604, colors as in C).

A, B. Smaller chromosomes are hotter for crossing over (A, centimorgans (cM) per kb; data of Mancera et al., 2008) and DSBs (B).
C, D. Telomere-proximal and pericentric DSB suppression. Points are oligo densities in 500 bp segments averaged across all 32 chromosome arms. Dashed line, genome average; magenta line, smoothed fit (lowess); shading, DSB suppression zones.
E. Multiple levels of spatial organization of the DSB landscape. Oligo distributions on Chr V were smoothed with different sized Hann windows. Dashed lines, genome average.
F. Correlation (Pearson's r) between Spo11 oligo density and GC content or binding of chromosome structure proteins, analyzed in variable sized windows. Rec8 data were from 2 hr in meiosis (Kugou et al., 2009); others were from vegetative cells (Lindroos et al., 2006; D'Ambrosio et al., 2008). See Supplemental Experimental Procedures.

A. Stereotypical chromatin structure of most yeast promoters.
B. Spo11 oligos cluster in promoter NDRs. Normalized nucleosome occupancy is the number of sequence reads covering a base pair divided by genome average coverage (Kaplan et al., 2009).
C. Genome average nucleosome occupancy and Spo11 oligo density around all annotated TSS and separately for TATA-containing and TATA-less genes.
D. Hotspots are nucleosome depleted (n=3600, omitting exogenous hotspots).
E. DSB activity is not correlated with degree of nucleosome depletion at hotspot centers. Hotspots were divided into quintiles by oligo count.
F. Low nucleosome occupancy is not sufficient for strong DSB activity. Mean profiles are shown for 3′ ends of convergent genes.
Spo11 oligos in C, D, F were smoothed with a 75 bp Hann window.

A. Schematic of a Spo11 DSB. Staggered cuts (blue arrows) by a Spo11 dimer generate a 2-nt 5′ overhang, the middle of which is a two-fold rotational symmetry axis. Nucleolytic cleavage (black arrow) forms the oligo 3′ end.
B. Nonrandom dinucleotide composition around Spo11 cleavage sites. At each position, deviation of dinucleotide frequencies from local average was summed (Supplemental Experimental Procedures). Gray line, deviation for a randomized sample of 50mers.
C. Biased DNA composition relative to predicted Spo11 binding. Dimeric structure is shown of a fragment of a Spo11 homolog, archaeal Top6A (Nichols et al., 1999), with monomers in green and blue, catalytic tyrosines in magenta. White bars indicate scissile phosphates in DNA docked on the dimer and color-coded by composition bias in B.
D. Spo11 preference at the scissile phosphate (dinucleotide indicated by the red circle).
E. Nonrandom dinucleotide composition around 3′ ends, as in B.
F, G. Nucleosomes and TFs are not a barrier to 3′ end formation. F, Spo11 oligos in an intergenic region. Cartoon depicts annotated nucleosomes (elipses), TSS (arrows), and Abf1 binding site. Graphs show 5′ and 3′ ends of oligos from top (red) and bottom (blue) strands. Nucleosome occupancy (3 hr) is in gray. G, Oligos around +1 nucleosomes (n=5,036). Genome averages were smoothed with 5-bp sliding window.