ATM/ATR-mediated phosphorylation of RAD18 is required for efficient S-phase progression and replication fork stability

During S phase, replicative DNA polymerases encountering DNA lesions may become stalled. Translesion synthesis (TLS) is a DNA damage tolerance mechanism whereby specialized DNA polymerases are recruited to sites of replication stalling to perform replicative bypass.RAD18, an E3 Ubiquitin ligase, has a central role in TLS. In response to replication fork stalling, RAD18 facilitates recruitment of TLS polymerases to sites of DNA damage and also mono-ubiquitinates PCNA. Checkpoint signaling, which is also activated by DNA damage, is widely hypothesized to coordinate cell cycle progression with DNA repair, thereby contributing to genome stability. However, putative mechanisms by which checkpoint signaling is integrated with RAD18-mediated TLS have not been identified. A recent proteomic screen identified Ser 403 of RAD18 as a potential substrate for the checkpoint kinases ATM and ATR. ATM/ATR-mediated phosphorylation of RAD18 provides a potential mechanism for regulation of TLS by checkpoint signaling. Therefore, the significance of ATM/ATR-mediated RAD18 Ser403 phosphorylation was investigated.Rad 18 Ser 403 phosphorylation occurred both during an unperturbed S phase and in response to genotoxin treatments that activate ATM and ATR. Ser403 phosphorylation of RAD18 did not affect its interactions with the TLS DNA polymerase Pol eta (Pol eta), or RAD6 (an E2 ubiquitin ligase). The recruitment of RAD18 to ssDNA at sites of DNA damage to repair foci in the nucleus was independent of Ser403 phosphorylation. However, failure to phosphorylate RAD18 at Ser403 led to replication inhibition and a DNA damage response, indicative of a role for ATR-dependent RAD18 Ser 403 phosphorylation in maintaining replication fork stability. Failure to phosphorylate RAD18 Ser403 in cells harboring a ubiquitination-resistant PCNA mutant also led to replication stress and a DNA damage response as measured by DNA damage marker upregulation. These results indicate that ATR signaling to RAD18 promotes replication fork stability independent of PCNA ubiquitination.These data demonstrate a novel mode of cross-talk between the ATR checkpoint signaling pathway and RAD18 and show that RAD18 phosphorylation by ATM/ATR kinases is important for preserving replication fork integrity, therefore defining a novel role for RAD18 during the cell cycle.