duced and analyzed for DSBs essentially as described . pat1+ strains were induced as described  without restoration of a nitrogen source at the beginning of the induction and with more vigorous agitation of cultures and use of baffled flasks. DNA from the times indicated was digested with NotI, separated on pulsed-field gels, and Southern hybridized using a 32P-labelled probe for the left end of the NotI-J fragment. The strains used were: (A) GP3718 (pat1–114 rad50S), (B) GP1979 (pat1–114 rad50+), (C) GP3124 (pat1+ rad50S), (D) GP3121 (pat1+ rad50+). W, well positions; 0, full-length NotI-J restriction fragment; 1, mbs1; and 2, mbs2. Quantitation of mbs1 and mbs2 at the time-point of maximal breakage is given on the right of each gel. Maximal DSB frequencies are greater in rad50S than rad50+ inductions because of DSB accumulation in rad50S. Maximal DSB frequencies are reduced in pat1+ compared to pat1–114 inductions due to greater asynchrony in the pat1+ inductions and a lower proportion of cells progressing through meiosis in that background.
(2.0 MB PDF)
Figure S10. Plots of Neighbor Dissimilarity (log2) against IP:WCE Hybridization Ratios for Each Probe of Chromosome I Using Data from the Haploid and Diploid Experiments (Strains GP6013 and GP6203)
Neighbor dissimilarity is expressed by calculating the absolute value of log2 (probe IP:WCE ratio/neighbor IP:WCE ratio), using both neighboring probes, and taking the lesser value.
(A) 0 h, haploid.
(B) Mean 5 h, haploid.
(C) 0 h, diploid.
(D) 5 h, diploid.
Note that high IP:WCE hybridization ratios in 0-h data are spikes; they have high dissimilarity relative to either neighboring probe. For these probes, dissimilarity rises with IP:WCE ratio, indicating that the neighboring probes have low (background) ratios, unrelated to that of the spikes. These spike probes are also seen in 5-h data, but another distribution of probes with high IP:WCE ratios and low neighbor dissimilarity is seen. These probes reflect genuine Rec12-enrichment with elevated IP:WCE ratios across several contiguous probes.
(8.2 MB PDF)
Figure S11. Plots of Neighbor Dissimilarity (log2) against IP:WCE Hybridization Ratios for Each Probe of Chromosome I using Data from Both Haploid and Diploid Experiments (Strains GP6013 and GP6203)
(A) 0 h, haploid.
(B) Mean 5 h, haploid.
(C) 0 h, diploid.
(D) 5 h, diploid.
Note that, in comparison to Figure S10, spikes have largely been removed but genuine Rec12-enrichment peaks remain.
(7.8 MB PDF)
Table S1. rec12-FLAG and rec12-HA Are Recombination Proficient
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Table S2. Correlation of DSB Hotspots and GO Classes
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Table S3. S. pombe Strains
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Table S4. Primers for Plasmid and Allele Construction and PCR Analysis
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Text S1. Construction of pJF32 (rec12-201::6His-2FLAG) and pJF41 (rec12-202::6His-2HA)
(23 KB DOC)
We are grateful to Yukang Lin for rec12 plasmids; Michael Lichten, Kim Lindstrom, Toshi Tsukiyama, and Iestyn Whitehouse for advice on immunoprecipations and their analysis by PCR; Chad Ellermeier for unpublished data; Jeff Delrow, Katherine Guthrie, and Harlan Robins for help with data analysis; and Jürg Kohli and Katja Ludin for exchange of data before publication. We thank Sue Amundsen, Luther Davis, and Walt Steiner for helpful comments on the manuscript.
GAC, RWH, SISG, and GRS conceived the experiments. RWH, HPC, and JAF performed the experiments. GAC and GRS analyzed the data and wrote the paper.
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