Rad52 Sumoylation Prevents the Toxicity of Unproductive Rad51 Filaments Independently of the Anti-Recombinase Srs2

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Rad52-L264P still interacts with RPA and Rad51.

<p>(A) To test the interaction with RPA, Rad52 or Rad52-L264P was immunoprecipitated with a rabbit anti-Rad52 polyclonal antibody from 1 mg of whole cell extracts (without DNAse treatment) prepared from <i>RAD52</i>, <i>rad52-L264P</i> or <i>rad52</i>Δ strains. To test the robustness of the interaction, increasing NaCl concentrations were added to the cell extracts. Proteins from the whole extracts (50 µg) and from the immunoprecipitated fractions were separated by SDS-PAGE and immunoblotted with rabbit anti-Rad52 polyclonal antibody or rabbit anti-RPA polyclonal antibody (allowing detection of the Rfa1 subunit of RPA). The signals corresponding to immunoprecipitated Rad52 or Rad52-L264P were quantified in three independent experiments and plotted as a fraction of the signal intensity measured in the 150 mM NaCl experiment. (B) To assess the interaction between Rad52-L264P and Rad51, Rad51 was immunoprecipitated from 1 mg of whole cell extracts (without DNAse treatment) from cells expressing Rad52-FLAG or Rad52-L264P-FLAG. The strength of the interaction was also evaluated against increasing NaCl concentrations. The proteins from whole cell extracts (50 µg) and from immunoprecipitated fractions were separated by SDS-PAGE and immunoblotted with rabbit anti-Rad51 polyclonal antibody and mouse anti-FLAG monoclonal antibody. The presence of Rad51 in the immunoprecipitated fraction cannot be detected to validate the efficiency of the immunoprecipitation because it migrates at the same level as the IgG anti-Rad51 used for the immunoprecipitation. However, the absence of Rad52 in the <i>rad51</i>Δ immunoprecipitate confirmed that the Rad52-FLAG signals observed are related to the Rad52-Rad51 interaction. The signals corresponding to immunoprecipitated Rad52 or Rad52-L264P were quantified in three independent experiments and plotted as in (A).</p

Over-expression of the <i>SIZ2</i> SUMO-ligase coding gene suppresses the MMS sensitivity of <i>srs2</i>Δ by stimulating Rad52 sumoylation.

<p>(A) Spot assay of haploid cells over-expressing <i>SIZ2</i>. Serial 10-fold dilutions were plated on minimal media lacking uracil with or without MMS. Strains of the indicated genotype were transformed with an empty vector or with the same plasmid containing the <i>SIZ2</i> gene. (B) Over-expression of <i>SIZ2</i> stimulates Rad52 sumoylation. Proteins conjugated with a His₇-SUMO radical were pull-down on Ni-NTA from 5 mg of extracts of <i>RAD52</i>-<i>FLAG</i> cells over-expressing His7-<i>SMT3</i>. Pull-downs were carried out from strains transformed with a <i>SIZ2</i>-containing multi-copy vector or with an empty vector. Cells treated with 0.3% of MMS were also tested as a positive control of sumoylation. <i>rad52-L264P-FLAG</i> strains were also subjected to pull-down analysis. Proteins from the whole extracts (3 µg) and from the pull-down fractions were separated by SDS-PAGE and immunoblotted with an anti-FLAG mouse monoclonal antibody.</p

ChIP analysis of Rad51 filament formation at a DSB created by the HO endonuclease in cells that express Rad52-FLAG, Rad52-L264P-FLAG or Rad52-SUMO-FLAG.

<p>The HO endonuclease was induced in WT or <i>srs2</i>Δ cells that express Rad52-FLAG, Rad52-L264P-FLAG or Rad52-SUMO-FLAG to create a single DSB that can be repaired by SSA. Samples were taken before induction and at 2, 4, 6 and 8 hours after galactose addition. Antibodies against the RPA complex, the FLAG epitope or Rad51 were used to precipitate protein-bound chromatin. Quantitative PCR was performed with primers located at 0.6 kb or 7.6 kb from the DSB site, using the immunoprecipitated chromatin (IP) and input DNA as template. As a control, primers specific for the <i>ARG5,6</i> locus were used. The relative enrichment represents the ratio of the PCR enrichment in the IP fraction to the input fraction. The median value of at least 3 experiments is shown and error bars represent the upper and lower values observed.</p

<i>rad52-L264P</i> suppresses mutations that are synthetically lethal with <i>srs2</i>Δ.

<p>(A) Tetrad analysis of crosses between haploid <i>rad52-L264P srs2</i>Δ strains and haploid mutants synthetically lethal with <i>srs2</i>Δ. Double mutant spores, which do not contain <i>rad52-L264P</i>, are indicated by white squares. The white circles mark triple mutants. (B) <i>siz2</i>Δ and <i>rad52-3KR</i> do not suppress the synthetic lethality of <i>srs2</i>Δ <i>sgs1</i>Δ and <i>srs2</i>Δ <i>rrm3</i>Δ mutants. In crosses involving <i>siz2</i>Δ, white squares display spores of <i>srs2</i>Δ <i>sgs1</i>Δ or <i>srs2</i>Δ <i>rrm3</i>Δ genotypes and white circles indicate triple mutants. To analyze the genetic interaction between <i>rad52-3KR</i> inserted at the <i>URA3</i> locus and the synthetically lethal <i>rrm3</i>Δ <i>srs2</i>Δ double mutant, diploids homozygous for <i>rad52</i>Δ were sporulated, in order to avoid the co-expression of <i>RAD52</i> and <i>rad52-3KR</i>. The white square indicates <i>srs2</i>Δ <i>rrm3</i>Δ <i>rad52</i>Δ triple mutants and white circles indicate unviable <i>srs2</i>Δ <i>rrm3</i>Δ <i>rad52</i>Δ <i>ura3</i>::<i>rad52-3KR</i> monosporic colonies.</p

Characterization of <i>rad52-L264P</i>, a suppressor of the MMS sensitivity of <i>srs2</i>Δ mutants that avoid the formation of toxic recombination intermediates.

<p>(A) Serial 10-fold dilutions of haploid strains with the indicated genotypes were plated onto rich media (YPD) containing different MMS concentrations. <i>rad52-L264P</i>* denotes the original isolated mutant strain and <i>rad52-L264P</i>** denotes a strain in which the <i>RAD52</i> gene was replaced by the mutant newly generated by directed mutagenesis. (B) Conservation of a motif comprising L264. The primary structure of Rad52 is schematized showing the conserved N-terminus moiety containing the major DNA binding and self-association domains (black, amino acids 1 to 179) as well as the C-terminus part (white, amino acids 180 to 471) containing the RPA (amino acids 275 to 278) and the Rad51 (amino acids 376 to 379) binding domains. The alignment of the Rad52 protein in Hemiascomycetes species shows the conservation of a domain containing the non-essential L264 residue and the QDDD residues essential for RPA binding and consequently for Rad52 mediator activity. The color code used in the alignment follows the default ClustalX color scheme as implemented in JalView (see Material and Methods). Cyan is for fully hydrophobic (I, L, V, M, F), turquoise for aromatic residues containing polar moieties (Y, H), green for small polar (T, S), purple for acidic (D, E), orange for glycine (G) and dark yellow for proline (P) residues.</p

Rad52 sumoylation prevents the toxicity of unproductive Rad51 filaments.

<p>(A) Schematic representation of the two kinds of toxic recombination intermediates eliminated by Srs2 in WT cells. We found that <i>srs2</i>Δ haploid cells sensitivity to DNA damage is related to recombination-deficient Rad51 nucleoprotein filaments. The toxicity of such filaments disappears in <i>rad52-L264P</i> srs2Δ cells, or alternatively, the intermediates themselves are not formed. However, <i>srs2</i>Δ cells sensitivity to DNA damage related with toxic intertwined HR intermediates cannot be suppressed by this allele. Unproductive Rad51 filaments can be formed after resection of a DSB located in a unique sequence in the genome. In a situation where homologous dsDNA cannot be found by the recombinase, Srs2 is essential to remove Rad51 filaments to allow alternative repair pathways such as SSA. Srs2 could also edit Rad51 filaments improperly nucleated by Rad52. In <i>srs2</i>Δ cells, nonrecombinogenic Rad51 filaments could also accumulate on ssDNAs generated from the uncoupling between the helicase complex opening replicative dsDNA and the DNA synthesis machinery in replicative mutants such as <i>mrc1</i>Δ. Finally, when a stable paranemic joint cannot be processed from a plectonemic joint because of mutations in genes involved in late recombination steps, Srs2 is necessary to address lesions to other DNA repair pathways. Intertwined recombination intermediates that occur between homologous chromosomes in diploid cells or between ectopic chromosomes in haploid cells cannot be suppressed by <i>rad52-L264P</i>. (B) Unproductive Rad51 filaments mediated by Rad52-SUMO are not toxic even in <i>srs2</i>Δ cells. When mediated by Rad52, Rad51 filaments that cannot complete strand invasion have to be removed by Srs2 in order to allow SSA or post-replication repair processes (PRR). Conversely, Rad52-SUMO (or Rad52-L264P) might lower (or shorten) Rad51 filaments. These modified mediators might also change Rad51 filament properties as indicated by Rad52 occupancy on Rad51 filaments. These changes might suppress Rad51 filaments toxicity, thereby bypassing the need for Srs2. Rad51 filaments might be removed by a Srs2-independent process.</p

<i>rad52-L264P</i> can only suppress <i>srs2</i>Δ deficiencies in the management of unproductive Rad51 filaments.

<p>Schematic representation and Southern blot analysis of the two HO-induced DSB repair systems involving SSA between two direct repeats 25 kb apart (A) and gene conversion between ectopic copies of <i>MAT</i> (D). The kinetic of repair in the SSA system after HO induction by addition of galactose to the medium was monitored by probing a Southern blot of <i>Kpn</i>I (K) digested genomic DNA of cells harvested at the indicated time with a PCR fragment complementary to the 3′end of <i>LEU2</i> (bold line). Quantification of the product band relative to the parental band (leu2::cs) measured at 24 hours is indicated (see Material and Methods for more information). To follow gene conversion of the <i>MAT</i>α allele after DSB induction, DNA was digested with <i>Cla</i>I (C1) and <i>Hind</i>III (H3), and probed with a <i>MAT</i> distal PCR fragment (bold line). The two possible outcomes, gene conversion associated with a CO or not (NCO), are indicated. The proportion of repaired products (NCO+CO) relative to the parental band (<i>MAT</i>α) and the proportion of CO among repair products measured at 10 hours are indicated (see Material and Methods for more information). (B and E) Cell viability after DSB formation in both assays. (C and F) Western blot analysis of Rad53 phosphorylation after HO induction in both systems.</p

Biochemical analysis of Rad52-L264P-mediated filaments.

<p>(<b>A</b>) The purity of recombinant Rad52 and Rad52-L264P (2 µg/each) was assessed by separation on 8% SDS-PAGE and staining with Coomassie blue. (B) Binding of Rad52-L264P to ssDNA. Protein titration reactions were performed by incubating 0.27 µM of a 62-nucleotides long Cy5-labeled ssDNA fragment with various amounts of Rad52-L264P at 37°C for 10 min (protein/bases: 1/10, 1/5, 1/2.5, 1/1.25, 1/0.6, 1/0.3, 1/0.15). Quantification of free ssDNA is shown. The data were fitted into a sigmoidal curve by using the PRISM software (GraphPad). (C) Rad52-L264P-mediated DNA annealing. Representative gels of Rad52 or Rad52-L264P-promoted DNA annealing are shown in the upper panel (Rad52/bases: 1/100, 1/42, 1/14, 1/6; same DNA as in (B) with reverse-complement, 340 nM each). The dsDNA/total DNA ratio at 10 min is shown in the lower panel. (D) RPA bound to ssDNA inhibits equally Rad52- and Rad52-L264P-catalyzed annealing reactions. Reactions were carried out with primers 25 and 26 (200 nM each, see Material and Methods) that were first incubated with 30 nM RPA (1/13 bases) at 30°C for 5 minutes, followed by addition of 40 nM Rad52 (1/10 bases). Self-annealing of the primers incubated without proteins and reactions performed without RPA or Rad52 are also shown. (E) Over-stimulation of DNA strand exchange by Rad52-L264P. <i>Upper panel</i>, diagram of the reaction substrates and products. <i>Middle panel</i>, ethidium bromide-stained DNA gel. As shown by the standard reaction (st), Rad51 efficiently catalyzes the formation of nicked circular products. Pre-bound RPA inhibits this reaction (line 2). Increasing amounts of Rad52 (lines 3–6, Rad52/bases: 1/55, 1/27, 1/18, 1/14) or Rad52-L264P (lines 7–10, Rad52/bases: 1/880, 1/220, 1/110, 1/55) overcome the inhibitory effect of pre-bound RPA. In line 1, only RPA was added to the reaction. <i>Lower panel</i>, the ratio of the nicked circular product over the sum of the linear dsDNA substrate and the nicked circular product is shown. (F) Salt titration of Rad51-Rad52/Rad52-L264P-ssDNA complex formation. The nucleoprotein complexes were assembled by incubating 0.8 µM Rad51 with 0.09 µM Rad52 or Rad52-L264P and 0.08 µM RPA pre-bound to 2.5 µM Cy5-labeled ssDNA (400 nucleotides) in the presence of the indicated NaCl concentrations at 37°C for 15 min. The Cy5 signals after nucleoprotein gel eletrophoresis are shown. Quantifications are shown below. Data were fitted into a third order polynomial curve. Western blot analysis of the gel using antibodies against Rad51 or Rad52 is also shown. Stars indicate signals corresponding to proteins not bound to ssDNA. (G) Transmission electron microscopy images of protein-DNA complexes showing the association of Rad52 with complete Rad51 filaments. Positive (left) and negative (right) staining images are shown for each type of filaments. The proportion of each type of complete Rad51 filaments formed by Rad52 or Rad52-L264P at different NaCl concentrations is shown and compared to a control reaction without Rad52. 100 molecules were examined in each experiment.</p

Rad52-L264P behaves like Rad52-SUMO.

<p>(A to E) Spot assay of haploid cells with the indicated genotype on rich medium (YPD) containing increasing MMS concentrations. Note that the deletion of the <i>SIZ2</i> gene (A) and the <i>rad52-3KR</i> allele (B) cannot suppress the MMS sensitivity of <i>srs2</i>Δ cells. Therefore, the MMS resistance of <i>rad52-L264P siz2</i>Δ <i>srs2</i>Δ and <i>rad52-3KR-L264P srs2</i>Δ cells is only related to <i>rad52-L264P</i>. (D) Note that the haploid strain spotted at the bottom contains both <i>rad52-3KR</i> and <i>RAD52</i> alleles.</p