Rad52 SUMOylation affects the efficiency of the DNA repair

Abstract

Homologous recombination (HR) plays a vital role in DNA metabolic processes including meiosis, DNA repair, DNA replication and rDNA homeostasis. HR defects can lead to pathological outcomes, including genetic diseases and cancer. Recent studies suggest that the post-translational modification by the small ubiquitin-like modifier (SUMO) protein plays an important role in mitotic and meiotic recombination. However, the precise role of SUMOylation during recombination is still unclear. Here, we characterize the effect of SUMOylation on the biochemical properties of the Saccharomyces cerevisiae recombination mediator protein Rad52. Interestingly, Rad52 SUMOylation is enhanced by single-stranded DNA, and we show that SUMOylation of Rad52 also inhibits its DNA binding and annealing activities. The biochemical effects of SUMO modification in vitro are accompanied by a shorter duration of spontaneous Rad52 foci in vivo and a shift in spontaneous mitotic recombination from single-strand annealing to gene conversion events in the SUMO-deficient Rad52 mutants. Taken together, our results highlight the importance of Rad52 SUMOylation as part of a 'quality control' mechanism regulating the efficiency of recombination and DNA repair.

Figure 1.

Purification of Rad52 species and SUMO machinery proteins. (A) Schematic representation of functional domain within S. cerevisiae Rad52 protein, including the positions of known SUMO acceptor lysines. The RPA interaction domain (black box, residues 308–311) and Rad51 interaction domain (grey box, residues 409–412) are indicated. (B) Purified SUMO machinery proteins: GST-tagged Aos1/Uba2 heterodimer (1 μg), (His)₆-tagged Ubc9 and Smt3 proteins (1 μg each) were run on a 15% SDS–PAGE and stained with Coomassie blue. (C) Purified (His)₆-tagged Rad52 species (1 μg each): Rad52, Rad52 (K43,44R), Rad52 (K253R) and Rad52 (K43,44,253R) were run on a 10% SDS–PAGE and stained with Coomassie blue.

Figure 2.

In vitro SUMOylation of Rad52 is stimulated by ssDNA. (A) The effect of ssDNA and dsDNA on Rad52 SUMOylation. In vitro SUMOylation assays were performed with recombinant Aos1/Uba2 (400 nM), Ubc9 (2.8 μM) and Smt3 (5.6 μM) in the presence or absence of ATP (2.5 mM), Rad52 (2.7 μM), 83-mer ss-DNA (100 μM nucleotides) or dsDNA as indicated. The reaction mixtures were incubated for 3 h at 30°C, stopped by adding 30 μl of SDS Laemmli buffer and analysed on 7% SDS–PAGE followed by silver staining. The hash symbol indicates high molecular poly-SUMOylated species of Rad52 and E1. The number at the bottom of the gel represents the ratio of modified and unmodified Rad52. SUMOylated Rad52 protein was confirmed by western blotting using anti-Smt3 (B) or anti-Rad52 (C) antibodies. (D) In vitro SUMOylation assay on Rad52 protein carried out without DNA (lanes 1 and 2) or with increasing amount of 83-mer ssDNA (15, 50, 100, 150, 300 μM nucleotides; lanes 3–7). The hash symbol indicates high molecular weight SUMOylated species. (E) Graphical representation of the gel in panel D, as a ratio of mono-SUMOylated versus non-modified Rad52. (F) The CD spectrum of 83-mer ssDNA (black), Rad52 (red) and the Rad52-ssDNA complex (white). The difference spectrum (green) represents values for Rad52 in the complex with DNA from which the spectrum of DNA has been subtracted. To form the complex, protein and DNA were incubated together for 10 min at 37°C prior to the analysis. The concentration of the protein was 3.87 μM and the concentration of DNA was 2.24 μM. The samples were measured at 10°C in 20 mM phosphate buffer containing 50 mM KCl, pH 7.5.

Figure 3.

Effect of accessibility of RPA- or Rad51-coated ssDNA on Rad52 SUMOylation. (A) RPA bound to ssDNA does not affect Rad52 SUMOylation. Increasing amounts of RPA protein (1.2, 3.5, 4.6, 5.9 and 8.3 μM) were pre-incubated with 83-mer ssDNA (100 μM nucleotides) for 10 min at 37°C and then mixed with E1 (400 nM), E2 (2.8 μM), Smt3 (5.6 μM), Rad52 (2.7 μM) and 2.5 mM ATP. After 3 h incubation at 30°C, the reactions were analysed by 7.5% SDS–PAGE and silver-stained. (B) The ratio of mono-SUMOylated versus non-modified Rad52 is presented by quantifying the corresponding bands in (A). (C) Rad51-coated ssDNA does not stimulate Rad52 SUMOylation. The reactions were carried out as in (A) except increasing amounts of Rad51 were used (3, 7, 14, 21, 27, 34, 41 μM). The reaction mixtures were stopped and analysed by 10% SDS–PAGE followed by silver staining. (D) Quantification of Rad52-Smt3 conjugate from C, as a ratio of mono-SUMOylated versus non-modified Rad52. The arrow indicates amount of Rad51 that fully coats ssDNA. The hash symbol indicates high molecular poly-SUMO chains of Rad52 and E1 proteins.

Figure 4.

Binding of Rad52 to ssDNA via C-terminal domain enhances the SUMOylation of lysine K253. (A) SUMOylation at lysine K253 is stimulated by ssDNA. The standard in vitro SUMOylation reaction was done with wild-type Rad52 (2.7 μM) or SUMO-deficient acceptor lysine Rad52 mutants: Rad52 (K43,44R), Rad52 (K253R) and Rad52 (K43,44,253R). (B) The C-terminal DNA binding domain of Rad52 is responsible for stimulation of SUMOylation. Rad52 protein and its fragments: Rad52 (N + M) (4.13 μM), Rad52 (M) containing GST-tag (3.52 μM) and Rad52 (M + C) (4.03 μM) were SUMOylated in vitro in the presence or absence of 83-mer ssDNA (100 μM nucleotides) and analysed. The asterisks indicate main SUMOylated Rad52 species. The hash symbol indicates high molecular poly-SUMO chains of Rad52 and E1 proteins.

Figure 5.

Rad52 SUMOylation affects its biochemical activities. (A) SUMOylation of Rad52 inhibits its binding to DNA. Increasing amounts of Rad52 or Smt3-Rad52 (20, 40, 80, 200 nM) were incubated with fluorescently labelled 49-mer ssDNA (0.49 µM nucleotides) at 37°C for 10 min. The reaction mixtures were resolved in 7.5% native polyacrylamide gels, and the DNA species were quantified using Quantity One software (Bio-Rad). (B) The results from (A) plotted. (C) SUMOylation of Rad52 inhibits its strand annealing activity. Labelled Oligo-1 (0.25 µM nucleotides) and Oligo-2 (0.25 µM nucleotides) were incubated separately with RPA (20 nM) for 3 min at 37°C. The annealing reactions were initiated by mixing RPA-coated oligonucleotides and Rad52 or Smt3-Rad52 proteins (0.7, 5, 10, 20 nM) and incubated at 37°C. After 8 min of incubation, 9 µl of the annealing reactions was removed and treated with 0.5% SDS, and 500 µg/ml proteinase K at 37°C for 10 min. The reaction mixtures were resolved in 12% native polyacrylamide gels. (D) The averaged values of results from three independent experiments are plotted.

Figure 6.

Rad52 oligomerization and interaction with RPA and Rad51 are not affected by its SUMOylation. (A) SUMOylation of Rad52 does not influence its oligomerization status. Rad52 and Smt3-Rad52 (2.3 µM) in 40 µl of buffer S were incubated with 4 µl of anti-FLAG agarose in 10 µl of buffer T containing 200 mM KCl for 30 min at 4°C. The beads were washed and treated with 25 µl of SDS Laemmli buffer to elute bound proteins. The supernatant (S) that contained unbound Rad52 or Smt3-Rad52 protein, wash (W) and the SDS eluate (E) (10 µl each) were analysed on 12% gel SDS–PAGE followed by staining with Coomassie Blue. The arrows and symbol hash denote anti-FLAG IgG and higher order SUMOylated species, respectively. (B) Analysis of Rad52 oligomerization status by gel filtration. Purified Rad52 or Smt3-Rad52 proteins (9 µM) in 200 µl of buffer S were filtered through a sephacryl S400 column. The indicated fractions were run on 10% SDS–PAGE followed by staining with Coomassie blue. The elution positions of the size markers are indicated: TG, thyroglobulin (669 kDa) and CAT, catalase (223 kDa). (C) SUMOylation of Rad52 does not affect its interaction with Rad51. Purified Rad52(M + C) or Smt3-Rad52(M + C) (22 µM) were mixed with Affi-Rad51 beads (4,6 µM Rad51) in 25 µl of buffer K and incubated for 30 min at 4°C. The beads were washed and treated with 25 µl of SDS Laemmli buffer to elute bound proteins. The supernatant that contained unbound Rad52 or Smt3-Rad52 protein, wash, and the SDS eluate (5 µl each) were analysed by SDS–PAGE in 12% gel followed by western blotting using anti-Rad52 antibody. (D) SUMOylation of Rad52 does not affect its interaction with RPA. The interaction with RPA was tested using purified Rad52 (M) or Smt3-Rad52 (M) (230 pmol) and Affi-RPA (1 µM RPA) beads pre-incubated with ssDNA (1 µg of ФX174) for 10 min at 37°C. Both mixtures were combined in 45 µl of buffer T followed by an incubation for 30 min at 4°C and then analysed as in panel C.

Figure 7.

Rad52-K43,44,253R foci exhibits shorter duration than wild-type. Time-lapse of spontaneous wild-type Rad52 and mutant Rad52-K43,44,253R foci. Strains W5094-1C (RAD52-YFP) and ML228 (rad52-K43,44,253R-YFP) were examined by fluorescence microscopy at 5 min intervals as described in the ‘Materials and Methods’ section. (A) Representative examples of time-lapse image sequences. Arrowheads mark Rad52 foci. (B) Quantitative analysis of time-lapse analysis. The number of cell cycles analysed is n = 173 for wild-type Rad52 and n=252 for the Rad52-K43,44,253R mutant protein. The class of cells for which no foci were observed amounted to 62 and 63% for RAD52 and rad52-K43,44,253R, respectively (not shown in the graph).