Figure 1.

RPA phosphorylation stimulates DNA synthesis during stress. (A) RPA2 phosphorylation mutants. The seven known human RPA2 phosphorylation sites are indicated by underlining, with the known or putative kinases shown. WT-RPA2, PIKK_A-RPA2 (double T21A/S33A mutation in the two PIKK sites), and Cdk_A-RPA2 (double S23A/S29A mutation in the two Cdk sites) variants were inducibly expressed from U2-OS stable clones. These three RPA2 variants and the PIKK_D (T21D/S33D mutation) and S4A/S8A mutants were transiently expressed in RPE cells. (B) Schematic of IdU/CldU labeling. (C) Representative fibers labeled with IdU (red) and CldU (green) from cells in which the endogenous RPA2 subunit was replaced with ectopic WT-RPA2, PIKK_A-RPA2, or Cdk_A-RPA2, as indicated. Images were resized to normalize IdU lengths, and the images were sorted so that molecules with the shortest CldU tracts were at the top and longest were at the bottom. For each set of tracts, the white line indicates the position of the IdU–CldU transition. These data indicate that cells replaced with WT-RPA2 have longer CldU tracts compared with cells replaced with PIKK_A- or Cdk_A-RPA2. Bar, 20 µm. (D) RPA2 phosphorylation significantly stimulates fork movement under replication stress conditions but not under unperturbed conditions. Quantitation of fork movement, expressed as the replication fork rate. The fork rate for cells replaced with WT-RPA2 was set at 1.5 kb/min. **, P < 0.01, relative to the fork rate of HU-treated cells replaced with WT RPA. Error bars indicate SEMs. (E) Schematic of [³H]TTP labeling involving either an early or late 90-min labeling period in the presence of 1 mM HU. (F) Mutation of RPA2 PIKK or Cdk phosphorylation sites primarily causes a general slowdown in replication fork movement. Endogenous RPA2 was replaced with ectopic WT-, PIKK_A-, or Cdk_A-RPA2. Two parallel batches of the appropriate replaced cell line were either (early) incubated in HU (15 min) and then HU and [³H]TTP for 90 min or (late) incubated in HU (105 min) and then HU and [³H]TTP for 90 min. The experiment was performed in triplicate. The amount of [³H]TTP incorporated into DNA in the early and late labeling periods was quantitated (see Materials and methods), and the value of [³H]TTP incorporated early/[³H]TTP incorporated late was plotted. The data are expressed as means ± SD.