FIGURE 5.
RPA-sedDNA complexes accumulate when gap-filling DNA synthesis is inhibited with HU/AraC. A, quiescent U2OS cells were left untreated or were pretreated for 30 min with 10 mm HU and 100 μm AraC before exposure to 10 J/m2 of UV. At the indicated time points, cells were harvested for isolation of sedDNAs, which were further immunoprecipitated with an anti-(6-4)PP antibody prior to radiolabeling, urea-PAGE, and phosphorimager analysis. B, whole cell lysates were prepared from quiescent U2OS cells with the indicated treatments and then subjected to immunoprecipitation with an anti-p62 (TFIIH) antibody. Protein content was analyzed by immunoblotting, and total sedDNA content was analyzed by radiolabeling, urea-PAGE, and phosphorimager analysis. Results from four independent experiments (average and S.D.) are graphed. C, whole cell lysates were prepared as in B but were subjected to immunoprecipitation with an anti-RPA32 (RPA) antibody.