FIGURE 2.
EXO1-dependent ATR Activation. A, a model system for excision repair-checkpoint coupling. ATR kinase reactions were carried out with ATR-ATRIP, TopBP1, RPA, and EXO1 as indicated. 0.6 ng (27 pm) single-stranded φX174 DNA (ssDNA), plasmid DNA (dsDNA), or gapped DNA was added to the reaction as indicated and incubated 20 min at 30 °C. Reactions were analyzed by immunoblotting for phospho-RPA2 (Ser33) and RPA1. B, EXO1-dependent phosphorylation of RPA2 (Ser33) and p53 (Ser18) in quiescent cells after UV damage. Serum-starved (quiescent) or asynchronously growing (proliferating) wild-type (WT) and Exo1−/− MEFs were exposed to 20 J/m2 of UVC light and harvested 1 h later. Cell lysates were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies.