DNA damage and secondary structures can act as potent obstacles to the replication machinery. Persistent stalling events lead to genomic instability and therefore numerous cellular tolerance mechanisms exist to complete genome duplication in the presence of such impediments. In addition to translesion synthesis (TLS) polymerases, eukaryotic cells contain a multi-functional
replicative enzyme called Primase-Polymerase (PrimPol) that is capable of directly bypassing DNA damage by TLS, as well as repriming replication restart downstream of lesions and secondary structures. Here, we report that PrimPol is recruited to re-prime stalled replication through its interaction with RPA. Using crystallographic and biophysical approaches, we identify that PrimPol
possesses two RPA-interacting motifs and identify the key residues required for these interactions. We demonstrate that one of these motifs is critical for PrimPol’s recruitment to stalled replication forks in vivo thus facilitating its role in re-priming DNA synthesis. In addition, biochemical analysis reveals that RPA serves to stimulate the primase activity of PrimPol. Together, these findings
provide unprecedented molecular insights into PrimPol’s mode of recruitment to stalled forks that enables it to efficiently re-prime the restart of DNA replication.