The role of RPA2 phosphorylation in homologous recombination in response to replication arrest

Abstract

Failure to reactivate stalled or collapsed DNA replication forks is a potential source of genomic instability. Homologous recombination (HR) is a major mechanism for repairing the DNA damage resulting from replication arrest. The single-strand DNA (ssDNA)-binding protein, replication protein A (RPA), plays a major role in multiple processes of DNA metabolism. However, the role of RPA2 hyperphosphorylation, which occurs in response to DNA damage, had been unclear. Here, we show that hyperphosphorylated RPA2 associates with ssDNA and recombinase protein Rad51 in response to replication arrest by hydroxyurea (HU) treatment. In addition, RPA2 hyperphosphorylation is critical for Rad51 recruitment and HR-mediated repair following HU. However, RPA2 hyperphosphorylation is not essential for both ionizing radiation (IR)-induced Rad51 foci formation and I-Sce-I endonuclease-stimulated HR. Moreover, we show that expression of a phosphorylation-deficient mutant of RPA2 leads to increased chromosomal aberrations following HU treatment but not after exposure to IR. Finally, we demonstrate that loss of RPA2 hyperphosphorylation results in a loss of viability when cells are confronted with replication stress whereas cells expressing hyperphosphorylation-defective RPA2 or wild-type RPA2 have a similar sensitivity to IR. Thus, our data suggest that RPA2 hyperphosphorylation plays a critical role in maintenance of genomic stability and cell survival after a DNA replication block via promotion of HR.

Fig. 1.

Hyperphosphorylation of RPA2 predominately occurs in response to replication arrest. (A) Whole cell lysates were isolated from MCF7 cells that were mock treated or exposed to replication inhibitor, HU (2 mM, 4 h) or Thy (2 mM, 48 h) and immunoblotted with an antibody specific to phosphorylated RPA2 (RPA2-p4/8, first panel) or RPA2 (Abcam, second panel). The same blot membrane from Figure 1A was subsequently reprobed with β-actin for loading control. (B) Whole cell lysates were isolated from MCF7 cells with or without IR treatment (8, 20 or 40 Gy, 6 h after) and then immunoblotted with antibodies against phosphorylated RPA2, RPA2 or β-actin. (C) The neutral comet assay of genomic DNA of MCF7 cells treated with replication inhibitor or IR. The doses used for HU and Thy are same as Figure 1A. The number of cells in each treatment group was 150. Results were expressed as Olive moment. The amount of breaks is correlated with the tail moment values (38).

Fig. 2.

Hyperphosphorylated RPA2 colocalizes at the ssDNA region in response to stalled/collapsed DNA replication forks. (A) HU treatment results in ssDNA accumulation. The protocol for ssDNA detection has been described in a previous publication (35). In brief, the cells were grown in the presence of bromodeoxyuridine (BrdU; 10 μg/ml; Invitrogen) for 24 h. Cells with BrdU labeling were treated with 2 mM of HU or left untreated and 6 h later, the cells were fixed. After fixation, the cells will be blocked and stained with anti-BrdU mouse monoclonal antibody clone B44 (BD Biosciences, San Jose, CA) antibody. Then, the samples are incubated with an Alexa Fluor 488-conjugated goat anti-mouse antibody. Cells were scored positive when 10 nuclear foci were visible. Quantitative analysis for ssDNA foci in response to HU was shown in Figure 2A. (B) Colocalization of phosphorylated RPA2 with ssDNA. MCF7 cells are cultured 24 h in the presence of BrdU. Cells with BrdU labeling were treated with 2 mM of HU and 6 h later, the cells were fixed. The fixed cells were costained by RPA2-p-4/8 antibody and anti-BrdU antibody. Representative phosphorylated RPA2 foci and ssDNA foci in response to HU were shown in Figure 2B. (C) The appearance of phosphorylated RPA2 foci occurs later than RPA2 foci after HU treatment. Cells in exponential growth phase were treated with HU (2 mM), fixed at indicated time points and costained with anti-RPA2 (Ab-2) and anti-phosphorylated RPA2 antibody (RPA2-p4/8). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue).

Fig. 3.

RPA2 hyperphosphorylation is required for Rad51 recruitment after HU treatment. (A) Phosphorylated RPA2 is colocalized with Rad51 after HU treatment. Exponentially growing MCF7 cells were treated with HU and fixed at 4 h after treatment. Cells were costained by RPA2-p4/8 and anti-Rad51 antibodies. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). (B) Schematic representation of RPA2 depicting phosphorylation sites mutant in the N-terminus of the protein. (C) Generation of cell lines expressing RPA2-WT or RPA2-A with endogenous RPA2 depletion is described in Materials and Methods. Whole cell extracts were immunoblotted with an anti-RPA2 antibody. (D) Hyperphosphorylation of RPA2 is required for Rad51 foci formation after HU treatment but not essential for IR induced Rad51 foci formation. After 18 h treatment with 2 mM HU or 8 h after 8 Gy IR, cells depleted of endogenous RPA2 and expressing RPA2-WT or RPA2-A were fixed and processed for immunocytochemistry with an anti-Rad51 antibody. Cells were scored positive when at least 10 nuclear foci were visible. Data shown are averages from three independent experiments (T-test, P < 0.01 for HU treatment; P > 0.05 for IR treatment). (E) Representative Rad51 foci in cells expressing RPA2-WT or RPA2-A. Representative Rad51 foci with HU treatment are shown in Figure 3E (upper panel). Representative Rad51 foci after IR treatment are shown in Figure 3E (bottom panel).

Fig. 4.

RPA2 hyperphosphorylation is required for the HR stimulated by HU but not for HR induced by I-Sce-I. (A) RPA2 hyperphosphorylation is specifically critical for HR after replication arrest. The frequencies of HR were calculated in cells expressing RPA2-WT or RPA2-A after HU treatment by flow cytometry. Results are means from three independent experiments with standard deviation. (B) Cells expressing phosphorylation-defective RPA2 show similar HR frequency induced by I-Sce-I expression in comparison to cells with wild-type RPA2. The HR frequency was measured by flow cytometric analyses (see Materials and Methods) in cells expressing RPA2-WT or RPA2-A. Results are means from three independent experiments, with standard deviation. (C) Immunoblot analysis of RPA2 hyperphosphorylation expression in MCF7 cells with I-Sce-I plasmid transfection or HU and Thy. Cell lysates were harvested 24 h after I-Sce-I transfection or after HU and Thy treatment. The doses used for HU and Thy are same as Figure 1A. Proteins were resolved by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and visualized by immunoblot with anti-RPA2-p4/8 antibody or anti-RPA2 antibody (Abcam). (D) Foci of phosphorylated RPA2 in cells with I-Sce-I transfection or HU treatment. The cells were fixed 24 h after I-Sce-I transfection or 4 h after HU treatment. Fixed cells were stained by a specific anti-RPA2 phosphorylation antibody and anti-RPA2-p4/8 antibody. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue).

Fig. 5.

RPA2 hyperphosphorylation is required for maintenance of genomic stability. (A) Frequencies of HU-induced chromosome aberrations are indicated. The data shown is from one of two independent experiments. Chid and Chme represent chromatid and chromosome, respectively. Fifty metaphases for each sample were analyzed. (B) Representative metaphase spreads in cells treated with 1 mM HU for 6 h and incubated in fresh medium for 24 h. Florescence in situ hybridization using telomeric and centromeric probes reveals the red/pink and green color. Chromatid-type aberrations indicated in the white arrow. Chromosome-type aberrations indicated in the yellow arrow. (C) Frequencies of HU-induced radial structures are indicated. Fifty metaphases for each sample were analyzed. The data shown is from one of two typical independent experiments. (D) A typical radical structure induced by HU from cells expressing RPA2-A. Y-shaped tri-radial chromosomes and star-shaped quadri-radial chromosomes were indicated by arrowheads. (E) Frequencies of IR-induced chromosome aberration are indicated. Fifty metaphases for each sample were analyzed. The data shown is from one of two typical independent experiments.

Fig. 6.

RPA2 hyperphosphorylation is critical for cell survival in response to HU but not IR. Clonogenic survival in MCF7 cells expressing RPA2-WT or RPA2-A after HU treatment (A) or exposure to IR (B). The cells expressing RPA2-WT or RPA2-A were treated with HU or IR at the indicated doses. The plotted points are on the averages of three to four experiments with standard deviations.