Generation of DSB maps at nearly single nucleotide resolution by Rec12-oligo mapping. (A) Pathway of meiotic DSB and Rec12-oligo formation. (B) Agreement between methods for mapping DSBs. Microarray hybridization of Rec12-DNA complexes (right axis, median-normalized IP/Input; data from Fowler et al. 2013), and uniquely mapped Rec12 oligos (left axis; offset in steps of 0.5 reads per million [RPM]/bp, smoothed with a 1-kb Hann window) are shown as an example region. An artifactual sequence pile-up at a single base pair at ∼710 kb in the 454 data set is not shown. Zoomed plots are provided in the bottom panels to show dispersed Rec12 oligos across the indicated subintervals of a DSB “cold” region. Orange arrows indicate the open reading frames of protein-coding genes. (C) Length distributions of Rec12 oligos. Oligo sequences were mapped to the genome and the distribution of alignment lengths was plotted according to mapping status (top). The sequence length distribution correlates with the sizes of DNA molecules purified from meiotic cultures and resolved by denaturing PAGE (bottom autoradiograph of duplicate lanes and trace). Note that the oligos migrate more slowly on the gel than expected due to residual amino acid(s) remaining on the 5′ end of each oligo after proteolysis and the addition of labeled nucleotides. (D) Agreement between high-resolution comparisons of breakage at the mbs1 hotspot (Southern blot of MluI-digested meiotic DNA from a rad50S induction at top; black line is the signal density trace) and Rec12-oligos (smoothed with a 501-bp Hann window). Orange boxes indicate 5′ portions of divergently transcribed flanking genes. (E) Quantitative correlation between Rec12 oligos and Rec12 ChIP-chip. Rec12 oligos were summed at hotspots determined by ChIP-chip (n = 288) and compared with the integrated microarray signal (Fowler et al. 2013). The trend is slightly nonlinear, likely because of higher microarray background. See Supplemental Figure S1 for other analyses and validation of Rec12-oligo distributions.
