1. Monitoring Homology Search during DNA Double-Strand Break Repair In Vivo 
Jörg Renkawitz, Claudio A. Lademann, Marian Kalocsay, Stefan Jentsch 
Molecular Cell 
Volume 50, Issue 2, Pages (April 2013)
DOI: /j.molcel
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2. Molecular Cell 2013 50, 261-272DOI: (10.1016/j.molcel.2013.02.020)
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3. Figure 1
Broad Distribution of Rad51 from the DSB over the Broken Chromosome
(A) (Top) Map of chromosome III of a donor-deficient MATα strain depicting the MATα locus (where a DSB can be induced by HO endonuclease), deleted HML and HMR sequences, recombination enhancer (RE), and centromere (dot). (Bottom) Rad51-directed ChIP at 0.2, 6, 54, and 75 kb away from the DSB. Rad51 enrichments are shown for the donor-deficient MATα WT strain and mutant derivatives deficient in Rad52, Rad55, and Rad57.
(B) Rad51-directed ChIP-chip 5 hr after HO expression using strains bearing single HO-cleavage sites (red arrows) either on chromosome I or chromosome IV. In addition to the symmetric Rad51 distribution surrounding the DSB on the effected chromosomes, small Rad51 enrichments are detectable around centromeres (blue dots) of all chromosomes and on the left arm of chromosome III, peaking at the RE element. Single spikes in the ChIP-chip data correspond to single oligonucleotides and are hybridization artifacts.
Data are depicted on a log2 scale. ChIP data are depicted as the mean ± SEM of IP/input ratio normalized to a control locus (chromosome X) and to the 0 hr time point. ChIP-chip data represent the mean of two experiments. See also Figure S1 and the Supplemental Experimental Procedures.
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4. Figure 2 Rad51 ChIP Signals Distribute beyond Regions of ssDNA
(A) ChIP-chip of Rad51 and RPA (Rfa1-myc; see Figure S2B for control using antibodies directed against untagged RPA) obtained in the strain of Figure 1B (single HO-cleavage site on chromosome IV). Profiles for a region of chromosome IV (200–800 kb) are shown before (0 hr) and 2 and 5 hr after HO induction.
(B) (Top) Map of chromosome III of donor-deficient MATα (as in Figure 1A). (Bottom) Time-resolved ChIP-chip profiles of Rad51, Rad52, and RPA (Rfa1-myc) for chromosome III obtained 1, 2, and 5 hr after HO induction. Asterisks denote experimental Rad52 and RPA peaks, which resulted from the use of tiling arrays that (unlike the strains used for ChIP) harbored HML and HMR sequences.
Gaps in ChIP-chip data correspond to repetitive DNA. Data (mean of two experiments) are depicted on a log2 scale. See Figure S2 for additional information.
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5. Figure 3
Rad51 ChIP Signal Levels Correlate with Yeast Mating-Type Donor Preference and Recombination Competence
(A–C) (Top) Shown are maps of chromosome III of donor-deficient MATα, MATa, and MATa ΔRE (deleted for RE) strains and known donor preferences (green arrows [Wu and Haber, 1996]). (Bottom) Shown are Rad51-directed ChIP-chip profiles (data representation as in Figure 2B) of these strains obtained 1, 2, and 5 hr after HO induction. Rad51-directed ChIP-chip data of (A) are identical to those of Figure 2B and are used for comparison. Positions of the DSB (red dashed lines) and RE (blue dashed lines) are indicated. Gaps in the profiles correspond to repetitive DNA.
(D) Diagram of Rad51-directed ChIP-chip profiles as in (B), but with a donor-proficient strain bearing intact HML (HML itself is not present on this array because of homology to MAT). Profiles depict early (30 min, 45 min, and 1 hr) and late (2 hr and 5 hr) time points after transient HO induction for 1 hr.
(E) (Top) Maps of regions of chromosome IV bearing a single HO-cleavage site (with flanking GFP-derived sequences) at 491 kb and homologous donor sequences positioned at different sites (625, 795, and 820 kb). (Bottom) Rad51-directed ChIP-chip profile in a corresponding donor-deficient strain (data from Figure 2A), underscoring Rad51 enrichment at position 625 kb in comparison to lesser enrichments at the two more distant donor homologies.
(F) Measurement of repair (see main text and the Supplemental Experimental Procedures) in the donor-proficient strains depicted in (E), 2 and 5 hr after transient HO induction for 1 hr. Data are indicated as ratios of the recombination product compared to a control locus (chromosome X).
(G) Rad51-directed ChIP in the donor-proficient strains depicted in (E) at 5, 49, 134, 304, and 329 kb away from the DSB. Positions 134, 304, and 329 kb away from the DSB represent positions directly next to the integrated homologous donor sequences.
ChIP and ChIP-chip data are depicted on a log2 scale, and the recombination product in (F) is depicted on a linear scale. ChIP data are depicted as the mean ± SEM of IP/input ratio normalized to a control locus (chromosome X) and to the 0 hr time point. ChIP-chip data represent the mean of two experiments. See Figure S3 for additional information.
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6. Figure 4
Homology Search Depends on Rad54 and the D Loop-Forming Activity of Rad51
(A) Rfa1-myc-directed ChIP using donor-deficient MATa cells, highlighting the appearance of RPA in areas surrounding the RE. These RPA signals, but not RPA signals in vicinity of the DSB, depend on Rad51 and Rad55.
(B) Rad51-directed ChIP in donor-proficient MATa WT and rfa1-t11 (K45E) mutant strains, indicating reduction of Rad51 signals around the RE in the mutant.
(C) Rad51-directed ChIP in donor-deficient MATα WT and rad51II3A (R188A, K361A, K371A) mutant strains, indicating that DSB distant Rad51 signals are lost with the respective mutant.
(D) (Top) Rad51-directed ChIP-chip profile depicting chromosome III in MATα WT, Δrdh54, Δrad54, and Δrad54 Δrdh54 strains. DSB-distant signals depend on Rad54 and Rdh54. (Bottom) Analogous Rfa1-myc-directed ChIP-chip profile in a MATα Δrad54 Δrdh54 strain. RPA (Rfa1) distribution mirrors Rad51 distribution in the Δrad54 Δrdh54 double mutant 4 hr after HO induction.
ChIP and ChIP-chip data are depicted on a log2 scale, and ChIP data are depicted as the mean ± SEM of IP/input ratio normalized to a control locus (chromosome X) and to the 0 hr time point. Single, double, and triple asterisks indicate p < 0.1, p < 0.015, and p < , respectively (Kruskal-Wallis one-way analysis of variance). ChIP-chip data represent the mean of two experiments and are presented after subtracting values for 0 hr signals. See Figure S4 for additional information.
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7. Figure 5 γH2A Follows Homology Search
(A) Comparison of γH2A and Rad51 distribution in a strain bearing a single SceI cleavage site (red arrow) on chromosome IV. ChIP-chip profiles obtained with antibodies specific for γH2A (orange) and Rad51 (black, 5 hr time point represents same data as in Figure 1B). The main difference between the two profiles is observed in the area of ssDNA around the DSB.
(B) γH2A-directed ChIP-chip using a strain bearing a single SceI-cleavage site (red arrow) on chromosome IV. Profiles for all chromosomes are shown 5 hr after HO expression. γH2A accumulates mainly on the broken chromosome, similar to Rad51 (Figure 1B). Centromeres of all chromosomes are indicated as blue dots.
(C–E) Comparison of γH2A and Rad51 distribution during homology search in the three strains of Figures 3A–3C. Overlay of ChIP-chip profiles obtained with antibodies specific for γH2A (orange) and Rad51 (black).
Data are depicted on a log2 scale and represent the mean of two experiments. See Figure S5 for additional information.
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8. Figure 6 Nuclear Organization Influences Homology Search
(A) Rad51-directed ChIP-chip profile depicting chromosome III (left) and an area of chromosome V (right) in a donor-deficient MATa ΔRE strain, in which the RE element has been transplanted to chromosome V. Rad51 signals are enriched around the transplanted RE, showing that this element can guide homology search in trans.
(B) Rad51 and γH2A ChIP-chip signals before and 5 hr after DSB induction surrounding centromeres of yeast chromosomes V, XI, and XVI. Grouped in columns are all ChIP-chip signals within 20 kb windows centered around the corresponding centromere. Significant Rad51 signals are detectable around all centromeres when the DSB is induced by either HO or SceI on chromosome I or chromosome IV, respectively (upper two panels, see Figure S6C for the remaining centromeres). The signals are lost when the SceI-cleavage site on chromosome IV is moved to a centromere-distant position (compare second upper and middle panel). No Rad51 signals around centromeres can be detected via HO break induction at the MAT locus (second lowest panel). Similar to Rad51, γH2A is enriched around the centromeres (bottom panel). The mean of the entire genome is put to 0 (highlighted as a horizontal green line), and the standard deviation is plotted in red. Data correspond to Figure 1B, Figure 2A, Figure 5A, Figure 6C, and Figure S1G.
(C) Rad51-directed ChIP-chip profile of an area of chromosome IV in strains bearing a single SceI cleavage site either at a centromere-proximal (upper panel) or centromere-distant (lower panel) position. The position of the centromere is indicated by a blue dot.
(D) Repair of a DSB on chromosome IV by donor homology on chromosome VII. (Top) Schemes of chromosome VII of three strains that harbor a single HO-cleavage site on chromosome IV and identical donor homology on chromosome VII at positions 13, 63, and 331 kb left from CEN7 of chromosome VII. (Bottom left panel) Rad51 ChIP signals at donor integration sites obtained from a corresponding donor-deficient strain (shown as fold enrichments; data are from ChIP-chip analysis of Figure 1B, collected within 1 kb windows around the donor integration sites) show that the signals increase with proximity to centromere CEN7. The mean of the entire genome is put to 0, and the standard deviation is plotted in red. (Bottom right panel) Accumulation of a recombination product 2 and 5 hr following transient HO induction for 1 hr in the above-mentioned donor-proficient strains, using the same analysis system as in Figure 3F. Recombination efficiency correlates with centromere proximity and Rad51 occupancy. Data are depicted as the mean of three experiments ± SEM.
ChIP-chip data are depicted on a log2 scale and represent the mean of two experiments.
Molecular Cell  , DOI: ( /j.molcel )
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