1. Recruitment of the Recombinational Repair Machinery to a DNA Double-Strand Break in Yeast 
Branden Wolner, Stephen van Komen, Patrick Sung, Craig L. Peterson 
Molecular Cell 
Volume 12, Issue 1, Pages (July 2003)
DOI: /S (03)

2. Figure 1
Recruitment of Rad51p-13Myc and Rad52p to an Induced DSB In Vivo
(A) Double-strand break induction kinetics. Galactose (2%, w/v) was added to cells in mid-log phase in order to induce HO endonuclease expression. Genomic DNA was purified at various time points, and PCR was performed using primers that flank the HO cut site from 96 bp CEN proximal of the HO cut site to 91 bp CEN distal. Appearance of the DSB is detected as a loss of PCR product (labeled DSB in the upper panel). Primers specific to the PHO5 promoter region (Krebs et al., 2000) were included in the PCR as a control. DSB bands were quantified using a phosphorimager and the ImageQuant software, normalized to the PHO5 bands, and plotted as the percentage of starting signal lost (“% Cut”). The chart in the lower panel represents the averages of at least four experiments per time point.
(B) Representative gels of ChIP assays performed using the same extracts as in (A). This strain lacked homologous donors sequences and harbored a 13Myc-tagged allele of RAD51. ChIPs were performed as described in the Experimental Procedures. “Input” DNA represents 1% of the total DNA used for the immunoprecipitations. DNA purified from the immunoprecipitations was subjected to PCR using primers specific to the PHO5 promoter region, as well as the MAT-Y (from 78 bp to 361 bp CEN proximal of the HO cut site) and the MAT-Z (from 18 bp to 257 bp CEN distal of the HO cut site) regions of the MAT locus. Note that both the anti-Rad51p-13Myc and anti-Rad52p IPs were performed using the same extracts.
(C) The bands in (B) were quantified. For each locus, the amount of signal in the IP was divided by the amount of signal in the corresponding input and then normalized to the time zero value giving a “relative % IP.” The results from the anti-Rad51p-13Myc IPs are plotted in the upper chart and those from the anti-Rad52p IP in the lower chart (PHO5, diamonds; MAT-Y, squares; MAT-Z, triangles). These data are representative of at least three independent short time course experiments.
(D) Long time courses of anti-Rad51p-13Myc (upper) and anti-Rad52p (lower) ChIP assays. These curves represent averages of three experiments for Rad51p-13Myc and five experiments for Rad52p. Error bars represent one standard deviation from the mean. Data obtained for Rad51p and Rad51p-13Myc using a polyclonal anti-Rad51p antibody were qualitatively comparable (data not shown). Note that the Rad52p ChIP dataset includes experiments from untagged Rad51 strains.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2 Recruitment of Myc-Tagged Rad54p and Rad55p to an Induced DSB
(A) Representative gels of PCR products from Rad54p-13Myc (upper) and Rad55p-13Myc (lower) ChIP assays in strains that lack homologous donor sequences.
(B) The bands in (A) were quantified and plotted as described in Figure 1 (PHO5, diamonds; MAT-Y, squares; MAT-Z, triangles). These data are representative of three independent time courses each.
(C) Long time courses for Rad54p-13Myc (upper) and Rad55p-13Myc (lower) ChIP assays. These curves represent the averages of three experiments each. Error bars represent one standard deviation from the mean.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3 Recruitment of Rad Proteins in Switching Strains
ChIP experiments were performed exactly as in Figure 1 but using strains that contain homologous donor sequences (silent mating-type loci). In order to assure only one round of switching, HO expression was repressed after 30 min by addition of glucose (2% w/v) to the medium.
(A) Kinetics of DSB formation and strand invasion. The formation of the synaptic complex is illustrated in the schematic, and the locations of the primers used in these assays are shown. The “strand invasion” product is not detectable until the incoming strand is extended at least 30 bp into the Yα-specific region of HML. PCR products are labeled as: DSB, from 192 bp upstream to 428 bp downstream of the HO cut site; Strand Invasion, from 30 bp upstream of the HO recognition site in HML to 428 bp downstream of the HO site in MAT; Act1, 254 bp of the ACT1 ORF.
(B) Representative ChIP experiment using polyclonal antibodies to Rad51p and Rad52p. PCR products: MAT-Z, from 18 bp to 428 bp downstream of the HO site; HMLα, from 257 bp upstream of the HO recognition site at HML to 10 bp downstream. ENA1, 290 bp of the ENA1 UAS on chromosome 4.
(C) Raw data from (B) were quantified, and relative percent IP was normalized to the percent IP of the ENA1 UAS to control for immunoprecipitation efficiency as described in Experimental Procedures. These results are representative of three trials each.
(D) Representative data from ChIP experiments using anti-myc antibodies and extracts from strains harboring either a 13Myc-tagged RAD54 or 13Myc-tagged RAD55 allele. Data were quantified as in (C) and are representative of two experiments each.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4 ChIP Assays in rad Mutant Strains
(A) Each chip assay was repeated at least twice in a strain harboring a complete deletion of one of the RAD52 group genes. Relative percent IPs were calculated as in Figure 1. Only the results of MAT-Z PCRs (as in Figures 1 and 2) are shown. Wild-type curves (diamonds) are identical to those shown in Figures 1D and 2C. Deletion mutants used were: rad51Δ, squares; rad52Δ, triangles; rad54Δ, circles; rad55Δ, crosses. Curves from these mutants are representative of at least two experiments each and are not normalized to an internal control.
(B) ChIP assays performed with switching strains. Rad51p was immunoprecipitated using a polyclonal antibody and extracts from either RAD54 or rad54Δ strains. (MAT-Z, diamonds; HMLα, squares). These curves are representative of three experiments each and are normalized to the ENA1 UAS control.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5 Rad54 Possesses a Mediator Function
(A) Effect of order of addition of reaction components on D loop formation. Presynaptic filaments were formed with or without Rad54p as described in Experimental Procedures. Rad54p was added in the initial incubation or after addition of ssDNA as indicated above the panels. The locations of DNA reactants and reaction products are indicated.
(B) Graphical representation of the results in (A). (Results from [AI], triangles; results from [AII], circles).
(C) Rad54p (0.05–0.7 μM) was either added with Rad51p and RPA to the ssDNA template (open squares, 6 min time point; closed squares, 9 min time point) or added after a preincubation of the ssDNA template with Rad51p and RPA (open circles, 6 min time point; closed circles, 9 min time point) as described in (A).
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6 Rad54 Promotes Nucleation of Rad51 onto ssDNA
(A) (AI) shows the schematic for examining the contents of the presynaptic protein complexes formed on immobilized DNA templates. The magnetic beads preloaded with the F1b/φX hybrid were used to bind Rad51p, Rad54p, and RPA, or combinations of these proteins. (AII) DNA-bound proteins were stripped from the magnetic beads with SDS and analyzed by electrophoresis in a 10% SDS-PAGE gel and staining with Coomassie blue. Only the Rfa1p subunit of the heterotrimeric RPA is shown.
(B) (BI) Rad51p (2.3 μM) and increasing concentrations of RPA (0.3, 0.8, 1.2, 1.7, and 2.2 μM in lanes 2–6, respectively) were mixed with the immobilized φX (+) strand. (BII) Rad54p (0.8 μM) and increasing concentrations of RPA (0.3, 0.8, 1.7, and 2.2 μM in lanes 2–5, respectively) were mixed with the immobilized φX (+) strand DNA. Proteins were eluted from the magnetic beads and analyzed as described in (A). The results from (BI) and (BII) are graphed in (BIII).
(C) (CI) Rad51p (2.3 μM), RPA (1.7 μM), and increasing concentrations of Rad54p (0.15, 0.35, 0.6, and 0.8 μM in lanes 3–6, respectively) were mixed with immobilized φX (+) strand DNA. Proteins were eluted from the magnetic beads and analyzed as in (A). The results in (CI) are graphed in (CII).
(D) Diagrammatic summary of the results. Rad51p forms a nucleoprotein-filament on ssDNA (Di) but is excluded from the ssDNA template by RPA (Dii). Binding of Rad54p to the ssDNA is much less prone to competition by RPA (Diii). Coincubation of Rad51p and Rad54p results in targeting of the former to the ssDNA template (Div).
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7 Recruitment of Rfa1p to a DSB
(A) Representative data from an Rfa1p-13Myc ChIP experiment using RAD54 or rad54Δ strains that lacked homologous donors. Similar results were observed in three independent experiments. PCR products are as in Figure 1.
(B) The raw data in (A) were quantified and plotted as described for Figure 1 (PHO5, diamonds; MAT-Z, triangles).
Molecular Cell  , DOI: ( /S (03) )