Rad18-dependent SUMOylation of human specialized DNA polymerase eta is required to prevent under-replicated DNA.

Translesion polymerase eta (polη) was characterized for its ability to replicate ultraviolet-induced DNA lesions that stall replicative polymerases, a process promoted by Rad18-dependent PCNA mono-ubiquitination. Recent findings have shown that polη also acts at intrinsically difficult to replicate sequences. However, the molecular mechanisms that regulate its access to these loci remain elusive. Here, we uncover that polη travels with replication forks during unchallenged S phase and this requires its SUMOylation on K163. Abrogation of polη SUMOylation results in replication defects in response to mild replication stress, leading to chromosome fragments in mitosis and damage transmission to daughter cells. Rad18 plays a pivotal role, independently of its ubiquitin ligase activity, acting as a molecular bridge between polη and the PIAS1 SUMO ligase to promote polη SUMOylation. Our results provide the first evidence that SUMOylation represents a new way to target polη to replication forks, independent of the Rad18-mediated PCNA ubiquitination, thereby preventing under-replicated DNA.

Regulation of the specialized DNA polymerase eta: revisiting the biological relevance of its PCNA- and ubiquitin-binding motifs.

During translesion synthesis (TLS), low-fidelity polymerases of the Y-family polymerases bypass DNA damages that block the progression of conventional processive DNA polymerases, thereby allowing the completion of DNA replication. Among the TLS polymerases, DNA polymerase eta (polη) performs nucleotide incorporation past ultraviolet (UV) photoproducts and is deficient in cancer-prone xeroderma pigmentosum variant (XPV) syndrome. Upon UV irradiation, the DNA sliding clamp PCNA is monoubiquitylated on its conserved Lys-164. This event is considered to facilitate the TLS process in vivo since polη preferentially interacts with monoubiquitylated PCNA through its ubiquitin-binding domain (UBZ) as well as its PCNA interacting peptide (PIP)-box. However, recent observations questioned this model. Therefore, in this study, we re-examined the relative contribution of the regulatory UBZ and PIP domains of polη in response to UVC. We show that simultaneous invalidation of both motifs confers sensitivity to UVC, sensitization by low concentrations of caffeine, prolonged inhibition of DNA synthesis and persistent S phase checkpoint activation, all characteristic features of XPV cells. While each domain is essential for efficient accumulation of polη in replication factories, mutational inactivation of UBZ or PIP motif only confers a slight sensitivity to UVC indicating that, although informative, polη focus analysis is not a reliable tool to assess the polη's ability to function in TLS in vivo. Taken together, these data indicate that PIP and UBZ motifs are not required for recruitment but for retention of polη at sites of stalled replication forks. We propose that this is a way to ensure that a sufficient amount of the protein is available for its bypass function.