Replication Protein A from the S-Phase Checkpoint Dissociation of Radiation-induced Phosphorylation of Updated Version

that this phosphorylation event may contribute to the inhibition of DNA replication activity (12, 13). These studies, in conjunction with the observation that cell lines derived from AT patients lack the irradiation-induced S-phase arrest (i.e., exhibit RDS; Refs. 22 and 23) and are deficient in IR-induced RPA phosphorylation (13, 14), sug gested that RPA phosphorylation may play a functional role in the regulation of the S-phase checkpoint. There are, however, several observations that would argue against such a functional relationship between irradiation-induced RPA phos phorylation and the S-phase checkpoint. First, inhibition of DNA synthesis in response to irradiation occurs within 30 mm after treat ment (22, 23), whereas optimal RPA p34 phosphorylation is not detected until 1†" 2 h postirradiation (24). Second, such phosphoryla tion of RPA is difficult to detect until radiation doses as high as 50 Gy are used (14), whereas the S-phase checkpoint is measurable at much lower (i.e., 2 Gy) doses of IR. To further explore the relationship between IR-induced RPA p34 phosphorylation and the S-phase checkpoint, we used recently devel o_ human cell lines that express either DN or complementing fragments of the ATM gene product (25†" 27). Cells expressing the DN fragment lose the IR-induced S-phase checkpoint, whereas AT cells expressing the complementing fragment regain the ability to arrest replicative DNA synthesis in response to JR (27). We analyzed these cells with manipulated ATM function to ask whether IR-induced RPA phosphorylation could be uncoupled from the IR-induced S-phase checkpoint. Results from our studies demonstrate that IR-induced RPA p34 phosphorylation is neither necessary nor sufficient for the S-phase checkpoint. lines from normal individuals (GM637) and AT homozygotes (GM5849), and the RKO colorectal carcinoma cell line were grown as described previously (27). HeLa cells were grown in DMEM with 10% FCS at 37°C and 5% CO2. Cells were irradiated with a @ 37Cssource at a dose rate of approximately 90 rad/min.