DNA-PK-Dependent RPA2 Hyperphosphorylation Facilitates DNA Repair and Suppresses Sister Chromatid Exchange
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.