1. In Vivo Roles of Rad52, Rad54, and Rad55 Proteins in Rad51-Mediated Recombination 
Neal Sugawara, Xuan Wang, James E. Haber 
Molecular Cell 
Volume 12, Issue 1, Pages (July 2003)
DOI: /S (03)

2. Figure 1 Localization of Rad51p to a DSB Created by HO Endonuclease
A donorless strain, JKM139, incapable of repairing a DSB, was grown in YP lactate medium and induced to express HO endonuclease with 2% galactose. Samples were taken before induction and at 1, 2, 4, and 7 hr after addition of galactose. Polyclonal antibodies against Rad51p were used to precipitate Rad51p-bound chromatin.
(A) Map of MAT shows the locations of the HO cut site and the primers (arrows), 189 to 483 bp distal to the DSB, used to PCR-amplify DNA from the immunoprecipitated DNA. As controls, primers to the ARG5,6 locus were used to amplify DNA from the immunoprecipitated chromatin and input DNA. PCR amplified DNA was run on ethidium bromide-stained gels (reverse images are shown).
(B) Quantitated signals were graphed for the wild-type strain. IP represents the ratio of the Rad51p IP signal to input signal from an independent locus, ARG5,6 (see Experimental Procedures). Cells were harvested at 30 min intervals after HO induction.
(C) Rad51p localization to MAT was followed either in a donorless strain where the DSB cannot be repaired or in a switching strain where the DSB can be repaired by gene conversion. Error bars show one standard error of the mean.
(D) Whole-cell extracts were treated with anti-Rad51 antibody and with anti-Mif2 antibody to simultaneously precipitate both Rad51p-MAT and Mif2p-CEN3 complexes. PCR signals from the MAT distal site (above) were normalized to the PCR signals from CEN3 signal and plotted as a function of time.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2 Rad51p Localizes to Sites Near the DSB
Rad51p-bound chromatin was precipitated from HO-induced strains before induction and at 1, 2, 4, and 7 hr after induction. DNA was PCR amplified from sites located proximal and distal to the DSB and then quantitated and graphed as described in Figure 1 and in the Experimental Procedures. The DSB is shown at 0 bp. HO was induced in a (A) donorless wild-type strain, JKM139 (B) JKM139 + pRS316, a URA3 centromere-bearing plasmid as a control, and (C) strain tNS2048 containing the RAD51-overexpressing plasmid (JKM139 + pNSU256). (D) Whole-cell extracts from tNS2048 were treated with anti-Rfa1, and precipitated chromatin was used to amplify a sequence 9.5 kb proximal to the DSB.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3
Effect of rad52Δ, rad54Δ, and rad55Δ on the Localization of Rad51p to an Unrepairable DSB
An unrepairable DSB was created in rad52Δ, rad54Δ, and rad55Δ strains, and Rad51p-bound chromatin was immunoprecipitated using anti-Rad51 antibodies. (A) PCR-amplified DNA from the MAT locus was run on ethidium bromide-stained gels. Reverse images of representative gels are shown. DNA signals were quantitated as described in the Experimental Procedures for (B) rad52Δ (JKM166), (C) rad55Δ (YLS52), (D) rad55Δ (YLS52), and (E) rad54Δ (YLS42) strains.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4
Rad51p Recruitment to HML and MAT during DSB-Induced Gene Conversion
Strains carrying an HMLα donor (HMLα MATa hmrΔ::ADE1 ade3::GAL::HO) were treated with 2% galactose to induce HO endonuclease and then with 2% glucose after 1 hr to repress HO. Anti-Rad51 antibodies were used to immunoprecipitate Rad51p-bound chromatin.
(A) PCR-amplified DNA, 189 to 483 bp distal to the DSB (MAT) and 188 to 467 bp from the uncleaved HO recognition site at HML, were prepared as described in Figure 1. Reverse images are shown. Quantitated signals were plotted for the MAT and HML loci in a wild-type strain (JKM161). One standard error is plotted.
(B) Early time points were also examined at 15 min intervals in cells grown and induced at 20°C.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5
rad54 Mutants Are Unable to Carry Out Primer Extension at HML after Strand Invasion
(A) Input DNA from wild-type (JKM161) and rad54Δ (tNS2045) strains from the ChIP experiments in Figure 4 was used to PCR-amplify DNA using a unique primer distal to MAT and a primer within the Yα sequence from HML (White and Haber, 1990). PCR products from samples before and after HO induction were run on ethidium bromide-stained gels. Reverse images are shown.
(B) Southern blot illustrates the physical monitoring of a DSB-induced gene conversion event as a wild-type strain, JKM161, switches from MATa to MATα. DNA was extracted from a time course culture used in Figure 4, digested with StyI, and prepared for Southern analysis using the MAT-specific probe (black box).
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6
Effect of rad Mutations on Rad51p Localization at HML during DSB-Induced Gene Conversion
Strains were induced to undergo DSB-induced gene conversion with 2% galactose followed by 2% glucose to repress HO expression. Anti-Rad51 antibodies were used to immunoprecipitate Rad51p-bound chromatin. (A) PCR-amplified DNA, 189 to 483 bp distal to the DSB (MAT) and 188 to 467 bp from the uncleaved HO recognition site at HML, were prepared as described in Figure 1. Reverse images are shown. Quantitated signals were plotted for (B) MAT and (C) HML in a rad55Δ strain (tNS2042), and (D) MAT and (E) HML in a rad54Δ strain (tNS2045). Error bars represent one standard error of the mean.
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7
Models of Rad52p, Rad54p, and Rad55p Participation in Rad51p-Mediated Homologous Recombination
Following a DSB at MATa, each end is resected by 5′ to 3′ nucleases. In the wild-type cell, a Rad51p filament forms on ssDNA in the MAT-Z region (red) (top row) and then promotes strand invasion at HMLα (bottom row). The sequences at HML that are not homologous with MATa are shown (Yα [gray] and HML-distal [hatched]). In the absence of Rad52p, no filament forms. Without Rad55p, filament formation may be incomplete and interrupted, perhaps because Rad55p and Rad57p are needed to remove RPA or facilitate binding to regions of secondary structure. The Rad51p that is bound to ssDNA in the absence of Rad55p is unable to facilitate strand invasion. Without Rad54p, formation of a Rad51p-DNA filament is normal and strand invasion occurs, but only to the point where a paranemic joint is formed. Formation of an interwound (plectonemic) joint that would allow the synthesis of new DNA, copying Yα, requires Rad54p. Alternatively, Rad54p allows removal of Rad51p from the heteroduplex DNA permitting strand extension and subsequent recombination steps.
Molecular Cell  , DOI: ( /S (03) )