CDC7-mediated phosphorylation of RAD18 facilitates recruitment of DNA polymerase eta to stalled replication forks

The TransLesion Synthesis (TLS) pathway facilitates the bypass of nucleotide lesions during DNA replication by recruiting specialized polymerases to replicate across the lesions. RAD18 plays a central role in the TLS pathway both by chaperoning POLH, a specialized polymerase, to stalled replication forks and by mono-ubiquitinating the polymerase clamp PCNA. The CDC7/DBF4 complex is a cell cycle kinase that plays a key role in regulating the initiation of DNA replication. For example, it has been shown to phosphorylate members of the MCM helicase complex, thereby contributing to the signal to begin DNA replication. Genetic evidence from S. cerevisiae has shown that RAD18 and CDC7 are epistatic to one another in the TLS pathway. However, nothing else is known regarding the possible involvement of CDC7 in this essential damage tolerance pathway in yeast or in higher eukaryotes. Our data suggest that in the human system, CDC7 directly phosphorylates RAD18 and contributes to its regulation in the context of TLS. We have shown that CDC7/DBF4 phosphorylates RAD18 in vitro and we have identified the serines that are targeted by CDC7 both in vitro and in living cells.;We have found that UV irradiation induces an interaction between RAD18 and CDC7 that occurs through the C-terminus of RAD18. Ablation of CDC7 or mutation of its target serines on RAD18 inhibits the formation of UV-induced POLH foci and abrogates the interaction between RAD18 and POLH. Finally, a mutant form of RAD18 that is unable to be phosphorylated by CDC7 remains competent to mono-ubiquitinate PCNA when it is used to complement Rad18-/- cells, demonstrating a separation of the E3 ligase activity and chaperoning properties of RAD18. Taken together, these results suggest a model in which CDC7-mediated phosphorylation of RAD18 promotes the interaction between RAD18 and POLH. These results provide a mechanistic explanation for the genetic relationship observed in yeast and highlight a novel relationship in which a cell cycle kinase participates in the regulation of the TLS pathway.