Rad52 inactivation is synthetically lethal with BRCA2 deficiency

Abstract

Synthetic lethality is a powerful approach to study selective cell killing based on genotype. We show that loss of Rad52 function is synthetically lethal with breast cancer 2, early onset (BRCA2) deficiency, whereas there was no impact on cell growth and viability in BRCA2-complemented cells. The frequency of both spontaneous and double-strand break-induced homologous recombination and ionizing radiation-induced Rad51 foci decreased by 2-10 times when Rad52 was depleted in BRCA2-deficient cells, with little to no effect in BRCA2-complemented cells. The absence of both Rad52 and BRCA2 resulted in extensive chromosome aberrations, especially chromatid-type aberrations. Ionizing radiation-induced and S phase-associated Rad52-Rad51 foci form equally well in the presence or absence of BRCA2, indicating that Rad52 can respond to DNA double-strand breaks and replication stalling independently of BRCA2. Rad52 thus is an independent and alternative repair pathway of homologous recombination and a target for therapy in BRCA2-deficient cells.

Fig. 1.

Rad52 shows synthetic lethality with BRCA2. (A) Cellular proliferation in EUFA423-BRCA2 complemented cells transfected with sh-Rad52 or control shRNA. (Left) Cell number measuring started 5 d after transfection and 3 d after selection in hygromycin. Cells were counted on a daily basis for 7 d. (Right) Photomicrograph shows the cells in culture at day 4. (B) (Left) EUFA423 cell lines, transfected with either sh-Rad52 or control shRNA as in A. (Right) Photomicrograph at day 4. For Rad52-depleted cells, little cell growth and abortive cell divisions are seen. In A and B the data are the mean ± SE from three independent experiments. (C) MCF7 cells were transfected with shBRCA2 or shRad52 or control construct. Cell lysates of each sample were made from puromycin- or hygromycin-resistant colonies and were immunoblotted as shown. Greater than 90% depletion of the target protein was observed, without a significant effect on the nontarget protein. β-actin and Filamin were used as loading controls. (D) Plating efficiency (clonogenic survival) of MCF7 cells following relatively acute depletion of BRCA2, Rad52, or both proteins. Cells were seeded using in the appropriate selection medium to assure stable suppression of the target protein. The results shown are from three independent experiments; error bars indicate SEM.

Fig. 2.

Rad52 expression and function is independent of BRCA2. (A) Capan-1 and EUFA423 cells were transfected with a plasmid containing wild-type BRCA2 or with an empty vector and then were analyzed by Western blotting. Nuclear extracts were probed for BRCA2 (Ab-1) and whole-cell extracts for Rad52 (5H9). (B) Ionizing radiation-induced GFP-Rad52 foci were detected in the EUFA423 cell line with or without WT-BRCA2. EUFA423 cells were transiently transfected with GFP-Rad52, treated 48 h later with 10 Gy of ionizing radiation, and incubated for 8 h. The percentage of cells with Rad52 foci is shown (mean and SE from three independent experiments).There was no significant difference in Rad52 foci with or without BRCA2 (P = 0.69 without ionizing radiation; P = 0.55 with ionizing radiation). (C) Ionizing radiation-induced GFP-Rad52 foci in Capan-1 cells with or without WT-BRCA2, as in B. There was no significant difference in Rad52 foci with or without BRCA2 (P = 0.52 without ionizing radiation; P = 0.37 with ionizing radiation). (D) Capan-1 and EUFA423 cells were transiently transfected with GFP-Rad52 and then were incubated for 2 h in the presence of BrdU, followed by immunofluorescence using an antibody to BrdU. (E) The percentage of Capan-1 and EUFA423 cells (treated as in D) containing Rad52 foci that also colocalized with BrdU foci. Bars represent SEM, based on three independent experiments, with nonsignificant differences. (F) Capan-1 cells were transiently transfected with GFP-Rad52 and 48 h later were treated with 10 Gy of ionizing radiation or sham treated and were incubated for 8 h before fixation for immunofluorescence studies using RPA2 antibody and DAPI.

Fig. 3.

Requirement for Rad52 in Rad51-mediated HR in BRCA2-deficient cells. (A) EUFA423 cells containing the recombination substrate, pDR-GFP, were transiently transfected with the I-SceI restriction endonuclease plasmid or with vector alone and 48 h later were analyzed for the percentage of green fluorescent cells. (B) EUFA423/pDR-GFP cells were transiently cotransfected with a combination of BRCA2 plasmid and Rad52 siRNA as indicated and were incubated for 24 h. The cells then were transiently transfected with the I-SceI restriction endonuclease plasmid or with vector alone. After an additional 48 h, the cells were harvested for Western blotting using BRCA2 (Ab-2) and Rad52 (5H9). (C and D) The frequency of (C) spontaneous recombinations and (D) I-SceI break-induced recombination (HR) were observed by flow cytometry. The bars represent the results of three or more independent experiments; error bars show SEM. Differences between control siRNA and Rad52 siRNA in BRCA2-deficient cells are significant: P = 0.0019 for spontaneous recombination, and P = 0.0025 HR, using an unpaired t test on logarithmic transformed data. (E and F) Frequency of (E) spontaneous recombinations and (F) I-SceI break-induced HR of MCF7 cells depleted of Rad52 or BRCA2, neither, or both using shRNA. The mean and SEM of three independent experiments is shown. The reduction in spontaneous recombination and HR for Rad52 depletion in BRCA2-deficient cells is highly significant: P < 0.001.

Fig. 4.

Severe chromosomal fragility in BRCA2- and Rad52-deficient cells. EUFA423 cells were transiently transfected with Rad52 siRNA and incubated for 72 h. Then metaphase chromosome spreads were prepared for FISH analysis. Centromere-specific PNA probes were labeled with fluorescein isothiocyanate (green), and telomere-specific PNA probes were labeled with the fluorochrome Cy3 (red). Representative images are as follows: (A) Chromatid break (arrow); (B) triradial and (C) quadriradial structures; (D) telomere fusions (arrow); (E) Chromosome end association (arrow); (F) mitotic crossover between nonsister chromatids (arrows); (G) mitotic chiasma (in box); and (H) meiotic synapsis between nonsister chromatids (in box). (I) For ionizing radiation-induced chromosome aberrations, EUFA423 cells were treated initially with Rad52 siRNA, then were irradiated with 2 Gy, and were incubated for a further 24 h. Metaphase chromosome spreads were prepared and stained with DAPI. Chromosome aberrations (chromatid breaks, chromosome breaks, triradials, and quadriradials) are shown by the arrows.