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Figure 3


Fig. 3. NBS1 and ATR are required for RPA phosphorylation following HU-induced replicative stress. (A) NBS-ILB1 (NBS1), NBS-ILB1 (S343A), NBS-ILB1 (S278A/S343A) and NBS-ILB1 (LXIN, vector only) cells were synchronized with 6 µM aphidicolin for 16 hours. The medium was replaced with fresh medium for 3 hours before HU treatment. Following 3 hours of treatment with 2 mM HU, cells were harvested and chromatin fractions were prepared. Proteins were detected by immunoblotting with the corresponding antibodies. (B) Chromatin isolated from the A-T cell line AT22IJE-T (ATM+) and its isogenic derivative cell line expressing recombinant ATM (ATM–) showed little difference in RPA-chromatin binding or RPA phosphorylation. Similar to the A-T cell lines, U2OS cells expressing either wild-type ATR or kinase-dead ATR (ATR-kd) displayed similar RPA-chromatin binding following DNA damage. Distinct from A-T cells, ATR-kd expression in U2OS cells significantly decreased RPA hyperphosphorylation.