Figure 1.
DNA damage that results in DNA replication stalling induces H2AX and RPA2 phosphorylation.
(A) Treatment of either 60 J/m2 UV irradiation, 0.5 mM hydroxyurea (HU), 2.8 µM camptothecin (CPT), 0.01% methyl methane sulfonate (MMS), or 50 µM 4-nitroquinoline 1-oxide (4NQO) in human HEK293T cells caused H2AX phosphorylation (γH2AX) and RPA2 hyperphosphorylation, whereas 5 Gy of γ-irradiation did not. Cells were treated with the indicated damaging agents for four hours before harvest. (B) Slowly migrating forms of RPA2 are due to phosphorylation. Treatment with λ-phosphatase reduced the slowly migrating forms of RPA2 to the migration position of the unmodified form. (C) Treatment with 50 µM or 200 µM roscovitine for one hour before 60 J/m2 UV irradiation in HEK293T cells suppressed RPA2 hyperphosphorylation. Hyperphosphorylation, intermediate phosphorylation, and no phosphorylation of RPA2 are indicated as H, M, and B, respectively.
Figure 2.
DNA-PK phosphorylates residues S4, S8 in RPA2 in response to DNA damage.
(A) RPA2 hyperphosphorylation induced by UV irradiation is dependent on DNA-PK. Expression of ATR, ATM, DNA-PK, TEL2, or CHK1 were silenced by siRNA in HEK293T cells and RPA2 hyperphosphorylation in response to 60 J/m2 UV irradiation was monitored. (B) RPA2 hyperphosphorylation is also dependent on the DNA binding subunit of DNA-PK, Ku86. (C) DNA-PKcs-null HCT116 cells do not express DNA-PKcs. (D) DNA-PKcs-null (DNA-PK−/−) HCT116 cells do not show RPA2 hyperphosphorylation in response to UV irradiation or 4NQO treatment compared to the parental HCT116 cells (DNA-PK+/+). (E) DNA-PK−/− (MO59J) cells do not express ATM and DNA-PK. (F) DNA-PK−/− (MO59J) cells do not show RPA2 hyperphosphorylation in response to 4NQO treatment compared to a matched DNA-PK+/+ (MO59K) cell line. (G) Lymphocytes defective in ATR (Seckel) or ATM (ATM−/−) as well as wild type lymphocytes produce RPA2 hyperphosphorylation in response to 4NQO treatment. Hyperphosphorylation, intermediate phosphorylation, and no phosphorylation of RPA2 are indicated as H, M, and B, respectively. NC, non-targeting control siRNA.
Figure 3.
RPA2 hyperphosphorylations correspond to the level of DSBs.
(A) Kinetics of RPA2 phsophorylation and γH2AX are similar after UV treatment. HeLa cells were irradiated with 60 J/m2 UV and S4, S8 phosphorylation of RPA2 and γH2AX were monitored. (B) DNA DSBs are generated in a similar kinetics with RPA2 phosphorylation after UV treatment. DNA DSBs by TUNEL assay were measured after 60 J/m2 UV irradiation using In situ Cell Death Detection Kit (Roche). (C) S4, S8 phosphorylated RPA2 foci are co-localized with γH2AX foci in response to UV irradiation. HEK293T cells were stained with specific anti-γH2AX or anti-phospho-RPA2 (S4, S8) (phospho-RPA2 (S4, S8)) antibodies after UV irradiation. (D) S4, S8 phosphorylated RPA2 and γH2AX are enriched at sites of stalled replication. Stalled replication forks that were pulse-labeled with BrdU were then immunoprecipitated with an antibody recognizing BrdU after cross-linking. Proteins in the immunoprecipitate were examined with specific antibodies as indicated.
Figure 4.
S4, S8 phosphorylation in RPA2 delays mitotic entry and confers resistance to 4NQO.
(A) Scheme for HU treatment. (B) RPA2 S4A S8A cells enter mitosis more frequently after release from HU treatment. More than 50,000 cells were counted for each cell type. The average from three independent experiments with standard deviation is presented. (C) Silencing the expression of DNA-PKcs makes cells enter mitosis more frequently after release from HU treatment. More than 50,000 cells were counted for each cell type. The average from three independent experiments with standard deviation is reported. (D) Cells expressing the RPA2 S4A S8A mutant are more sensitive to 4NQO.
Figure 5.
S4, S8 phosphorylation of RPA2 inhibits homologous recombination.
(A) Cells expressing the RPA2-S4A S8A mutant protein generate more RAD51 foci than cells expressing wild type (WT)-RPA2. RAD51 foci were counted after cells were treated with 5 Gy of γ-irradiation with 6 hr of recovery. 275 cells of wild type (WT) and 323 cells of mutant (S4A, S8A) RPA2 were analyzed for RAD51 foci formation. (B) Cells expressing the RPA2 S4A, S8A mutant express more RAD51 than WT-RPA2 cells. RAD51 histograms were measured in cells treated with HU for 22 hr or mock treated and allowed to recover for 6 hr. Median numbers are provided. (C) The S4A, S8A mutations cause an increase in the UV-induced SCE rate in HEK293T cells (P<0.0001). Two independent experiments were performed and the average percentage of SCE with standard deviation is reported. A total 66 WT-RPA2 and 89 RPA2 S4A, S8A metaphases cells were counted.