Volume 13, Issue 3, Pages 389-401 (February 2004) Association of Mre11p with Double-Strand Break Sites during Yeast Meiosis  Valérie Borde, Waka Lin, Eugene Novikov, John H. Petrini, Michael Lichten, Alain Nicolas  Molecular Cell  Volume 13, Issue 3, Pages 389-401 (February 2004) DOI: 10.1016/S1097-2765(04)00034-6 Copyright © 2004 Cell Press Terms and Conditions

Figure 1 Transient Mre11p Binding to DSB Site in Wild-Type Cells (A) Map of the regions used for PCR detection of Mre11p binding. Chromosome III is depicted on top. Hatched boxes, DSB-rich domains; white boxes, DSB-cold domains (Baudat and Nicolas, 1997); circle, centromere. Horizontal arrows represent the ORF regions. Vertical arrows indicate DSBs in the promoter of YCR048W (∼9% of chromosomes) and weaker DSBs at neighboring sites (Baudat and Nicolas, 1997). Horizontal bars indicate the position of the PCR fragments for YCR048W (164 bp) and YCR013C (220 bp). (B) Mre11p ChIP from wild-type (ORD7339) cells at the times indicated in meiosis, detected by multiplex PCR with the primers specific for YCR013C and YCR048W. (C) Southern blot of StuI digested DNA from cells from the same sporulation as in (B), probed with YCR065W. The promoter site of YCR061W (arrow) was used for DSB quantification. (D) Graph showing DSB quantification (diamonds) and the ratio of YCR048W versus YCR013C PCR product obtained after ChIP with (closed squares) or without (open squares) Mre11p antibody. The experiment was performed independently three times and gave similar Mre11 ChIP enrichment profile, with a peak at 4 hr and maximum ratio varying from 3.5 to 9. One representative experiment is shown. (E) Cumulative curves of replication intermediate (triangles), Mre11p binding at YCR048W (squares), and DSBs (diamonds). The sum of intermediates was adjusted to 100%. For Mre11p binding, 1 was subtracted from the YCR048W/YCR013C ratios shown in (D). Closed circles, percent of nuclei that have passed MI division. Molecular Cell 2004 13, 389-401DOI: (10.1016/S1097-2765(04)00034-6) Copyright © 2004 Cell Press Terms and Conditions

Figure 2 Microarray Analysis of Mre11p and Spo11p Chromatin Association in sae2 Meiotic Cells (A) Partial image of a microarray hybridized with the labeled Mre11- or Spo11-HA3His6-immunoprecipitated DNA (Cy5, red color) and control WCE (Cy3, green color), from sae2 cells (ORD7311) at T = 6 hr. The red spot indicated by an arrow corresponds to the YGR176W ORF, strongly enriched in both immunoprecipitated samples. (B) Comparison of the distribution of ChIP/WCE ratios for Mre11p at T = 0 hr, Mre11p at T = 6 hr and Spo11-HA3His6 at T = 6 hr. The average hybridization ratios for each ORF were grouped into intervals of 0.05 log2 units, and the number of ORFs were counted in each interval. For comparison, the profile of ratio distribution from the 0 hr experiment is plotted in pink on the graphs from the 6 hr Mre11 and Spo11 experiments. The deviation of the curve to the right of the 0 hr curve represents the set of ratios of the preferentially immunoprecipitated targets. Molecular Cell 2004 13, 389-401DOI: (10.1016/S1097-2765(04)00034-6) Copyright © 2004 Cell Press Terms and Conditions

Figure 3 Genome-Wide Comparison of Mre11p ChIP versus Covalently Bound Spo11-HA3His6p (A) Comparative genomic map of Mre11p chromatin association (top) and Spo11-dependent DSB regions (bottom) for each chromosome (I to XVI) determined by genome-wide location analysis performed in sae2 diploid cells (ORD7311) at 6 hr in meiosis. Each vertical bar corresponds to one ORF. The average normalized ratio between immunoprecipitate versus WCE is indicated by a color code from green to red. ORFs are arranged following their respective position on each chromosome. Blue circles indicate the position of centromeres. (B) A magnified view of chromosome III is displayed for comparison of DSBs mapped by Southern blot analysis (Baudat and Nicolas, 1997) and by ChIP (Spo11-HA3His6) with Mre11p chromatin association regions. Position of centromere (blue circle) is indicated. Average ratios are indicated for each ORF median chromosomal location. Molecular Cell 2004 13, 389-401DOI: (10.1016/S1097-2765(04)00034-6) Copyright © 2004 Cell Press Terms and Conditions

Figure 4 DSB Formation Is Not Required for Mre11p Association with DSB Site spo11-Y135F (ORD7341) (A), spo11Δ (ORD3592) (B), or spo11-Y135F ndt80Δ (ORD7374) (C) ChIP with anti-Mre11 antibody at the indicated time in meiosis. PCR amplification as in Figure 1B. Graphs show the relative enrichment of YCR048W DSB site versus YCR013C control site upon Mre11p immunoprecipitation (closed squares). Open squares, minus antibody control. Closed circles, % cells that have passed the first meiotic division. The spo11-Y135F experiment was repeated independently twice and gave similar binding profiles, with a maximum enrichment value at 3 hr from 7.5 to 14. The spo11Δ experiment was done in duplicate. For spo11-Y135F ndt80, data are from a single experiment and gave similar binding profile to that seen in spo11-Y135F. Molecular Cell 2004 13, 389-401DOI: (10.1016/S1097-2765(04)00034-6) Copyright © 2004 Cell Press Terms and Conditions

Figure 5 Absence of Mre11p Binding at DSB Site in Several DSB-Deficient Mutants Formaldehyde-treated cells were lysed and immunoprecipitated with anti-Mre11 antibody at the indicated time in meiosis. Graphs for Mre11p enrichment ratio at YCR048W and nuclear divisions: legend as in Figure 4. (A) rec102Δ: ORD5854; rec103Δ: ORD7370; rec104Δ: ORD5853; rec114Δ: ORD7347; mer2Δ: ORD7348; mei4Δ: ORD7358. All experiments were done once except for rec102Δ and mei4Δ, which were done in duplicate. (B) Mutants affecting the Mre11p complex. mre11ΔC49 (ORD7343); rad50Δ (ORD7350), and xrs2Δ (ORD7359). mre11ΔC49 experiment was done twice; rad50Δ experiment was done in two independent experiments that gave similar results, showing a maximum Mre11 enrichment ratio at 3 hr of 3 and 9. xrs2Δ: a single experiment was done. Molecular Cell 2004 13, 389-401DOI: (10.1016/S1097-2765(04)00034-6) Copyright © 2004 Cell Press Terms and Conditions

Figure 6 Rad50 Association with DSB Site Depends on Mre11p and Xrs2p (A) Upper panel: DSB formation at YCR048W in the Rad50-Myc cells (ORD8216); Genomic DNA was cut by BglII. Lower panel: parallel Rad50-Myc ChIP-PCR (from the same time-course of formaldehyde-treated cells. The graph compares YCR048W DSB formation (diamonds) and relative Mre11p association with YCR048W (closed squares). Open squares, minus antibody control. Other legend as in Figure 4. (B) Upper panel: DSB formation at YCR048W in sae2 cells (ORD7398). DNA was cut with AseI. Lower panel: Rad50-Myc ChIP-PCR from the same formaldehyde-treated cells (ORD7398). The graph compares YCR048W DSB formation and Mre11 association as in (A). (C) Rad50-Myc ChIP enrichment ratio at YCR048W in mre11Δ (ORD8207) and xrs2Δ (ORD8220) meiotic cells. Molecular Cell 2004 13, 389-401DOI: (10.1016/S1097-2765(04)00034-6) Copyright © 2004 Cell Press Terms and Conditions

Figure 7 Accumulation of Mre11p at DSB Site in Cells that Accumulate Unprocessed DSBs (A) Mre11p ChIP-PCR of formaldehyde-treated sae2 cells (ORD7311). Plus and minus antibody samples are shown. (B) DSB formation at YCR048W in cells from the same meiotic time-course as in (A). (C) Comparison of YCR048W DSB formation (diamonds) and relative Mre11p association (closed squares). Open squares: minus antibody control. The experiment was performed independently four times and gave similar Mre11 ChIP enrichment profile, with a ratio at 6 hr varying from 26 to 77. (D) Mre11p ChIP from mre11-58S cells (ORD7353). (E) DSB analysis as in (B). (F) Comparison of DSB and relative association of Mre11p with the YCR048W region as in (C). The experiment was performed independently twice and gave similar Mre11 ChIP enrichment profile, with a ratio at 6 hr varying from 26 to 49. (G) Mre11p ChIP from dmc1Δ cells (ORD7354). (H) DSB analysis in the YCR065W region. (I) Comparison of DSB and relative association of Mre11p with the YCR048W region as in (C). Molecular Cell 2004 13, 389-401DOI: (10.1016/S1097-2765(04)00034-6) Copyright © 2004 Cell Press Terms and Conditions