The N-terminal tails of histones H3 and H4 and the acetylatable lysines of histone H4 confer resistance to the HO endonuclease. A. Sensitivity to prolonged exposure to HO endonuclease. S. cerevisiae strains RMY200 (wild type [WT]), RMY430 (H3 tailΔ), BAT039 (H4 tailΔ), FLY722 (H4K5,8,12,16R), FLY821 (H4K5,8,12R), and BAT038 (rad52Δ) containing the pGAL-HO-ADE2 plasmid were serially diluted onto plates containing increasing amounts of galactose (GAL) in order to induce increasing amounts of the HO endonuclease. B. Sensitivity to limited exposure to HO endonuclease. The viability of yeast strains used in panel A was determined after induction of the HO endonuclease for 0, 1, 2, or 3 h. The average viability ± standard deviation (error bar) of three independent ChIP experiments is shown. Viability at 0 h is normalized to 100 percent.

Levels of histone acetylation before, during, and after repair of a double-strand break by homologous recombination. A. Schematic of the mating-type loci in yeast. The locations of PCR primers used to quantify HO cleavage and repair are shown, where the reverse primer is specific to the MAT locus and the forward primers recognize the X sequence. If the mating type is a, then the PCR product is 1.0 kb, and if it is α, then the product is 1.1 kb. Also shown are the positions of primer pairs used to assay the amount of acetylation 0.6 kb and 2 kb away from the HO endonuclease site. Chrom.III, chromosome III. B. Cutting and repair of the HO lesion in wild-type yeast. HO endonuclease was induced at time zero in strain BAT009 (wild type) containing the plasmid pGAL-HO-URA3 by the addition of galactose and was repressed at 2 h by the addition of glucose. Samples were analyzed throughout the time course with the primers shown in panel A and control primers. The MATa and MATα products were quantified from the gel and were normalized to the control product. The amount of MAT product at time zero was normalized to 1. C. ChIP analysis of acetylated lysines on histones H4 0.6 kb and 2.0 kb away from the HO site. HO endonuclease was induced at time zero in strain BAT009 containing the plasmid pGAL-HO-URA3 by the addition of galactose and was repressed at 2 h by the addition of glucose as described above for panel B. Relative percent immunoprecipitation (IP) was quantitated by taking the ratio of the MAT product to the SMC2 control product. To obtain the IP/input values (y axis), the amount of immunoprecipitated DNA was divided by the amount of the input DNA. Values were normalized so that the IP/input ratio was equal to 1 at time zero. The averages and standard errors of the means (error bars) are plotted for at least six independent quantitative real-time PCR experiments from two independent ChIPs. The asterisks represent a significant P value where one asterisk represents a P value of 0.05 to 0.01, two asterisks 0.009 to 0.001, and three asterisks ≤0.0009. Black asterisks indicate a significant change from the previous time point, and small grey circles indicate a significant change from the value before HO induction at time zero. H4AcK5, H4 with lysine 5 acetylated; H3 C-term, H3 C terminus. D. ChIP analysis of histone H3 levels and acetylated lysines on histone H3, as described for panel C.

Gcn5, Esa1, Sir2, Hst1, and Rpd3 may act to modify histones during homologous recombination. A. Sensitivity to prolonged HO induction. Serial dilution analysis of strains BAT021 (wild type [WT]), BAT019 (gcn5Δ), BAT020 (rpd3Δ), and BAT022 (rad52Δ), containing plasmid pGAL-HO-URA3 onto plates with and without galactose (GAL). B. Sensitivity to transient induction of HO endonuclease. Viability analysis of the strains used in panel A following transient exposure to HO endonuclease performed as described in the legend to Fig. 1B. C. Gcn5 and Esa1 are recruited to the HO lesion. ChIP analysis of strains Z1466 (Gcn5-MYC), Z1346 (Esa1-Myc), and Z1256 (no tag) containing a pGAL-HO-URA3 plasmid where galactose was added after time zero and glucose was added after 2 h to induce and repress the HO endonuclease. Real-time PCR analysis and quantification was performed as described in the legend to Fig. 2 using primers 0.6 kb to the right of the HO site and an SMC2 internal primer pair for normalization. D. Sir2, Hst1, and Rpd3 are recruited to the HO lesion. ChIP analysis of strains BAT035 (Sir2-HA) and BAT009 (No tag), Z1346 (Hst1-MYC) and Z1256 (No tag), and Z1347 (Rpd3-MYC) and Z1256 (No tag) containing a pGAL-HO-URA3 plasmid as described for panel C.

Changes in histone H3 and H4 acetylation are triggered by homologous recombination. A. Cutting and repair of the HO site were measured when the HO endonuclease was induced at 0 h and repressed at 2 h in strain JKM179 (hml/hmrΔ) containing plasmid pGAL-HO-URA3. B. An H3 C-terminal (H3C-term) antibody was used to measure the relative levels of histone proteins 0.6 kb away from the HO site by ChIP, real-time PCR, and statistical analyses as described in the legend to Fig. 2. C. Levels of histone acetylation in strain JKM179 (hml/hmrΔ) containing plasmid pGAL-HO-URA3 at 0.6 kb from the HO site were measured as described for panel B and in the legend to Fig. 2. Significant changes are noted by small light grey circles as described in the legend to Fig. 2.

Changes in histone H3 and H4 acetylation are triggered by the Rad52 protein. A. Cutting and repair of the HO site in strain BAT022 (rad52Δ) containing plasmid pGAL-HO-URA3. B. Histone acetylation levels were measured in the same strain used in panel A as described in the legend to Fig. 2. Significant changes are noted by asterisks and small grey circles as described in the legend to Fig. 2.

Levels of histone acetylation before, during, and after repair of a double-strand break by homologous recombination at the homologous donor sequence. DNA from the ChIP analysis in Fig. 2 was reexamined using primers adjacent to the HO consensus sequence in the HMLα gene to measure histone acetylation levels. Significant changes are noted by asterisks and small light grey circles as described in the legend to Fig. 2.