DNA polymerase D temporarily connects primase to the CMG-like helicase before interacting with proliferating cell nuclear antigen

The eukaryotic replisome is comprised of three family-B DNA polymerases (Pol alpha, delta and epsilon). Pol alpha forms a stable complex with primase to synthesize short RNA-DNA primers, which are subsequently elongated by Pol delta and Pol epsilon in concert with proliferating cell nuclear antigen (PCNA). In some species of archaea, family-D DNA polymerase (PolD) is the only DNA polymerase essential for cell viability, raising the question of howit alone conducts the bulk of DNA synthesis. We used a hyperthermophilic archaeon, Thermococcus kodakarensis, to demonstrate that PolD connects primase to the archaeal replisome before interacting with PCNA. Whereas PolD stably connects primase to GINS, a component of CMG helicase, cryo-EM analysis indicated a highly flexible PolD-primase complex. A conserved hydrophobic motif at the C-terminus of the DP2 subunit of PolD, a PIP (PCNA-Interacting Peptide) motif, was critical for the interaction with primase. The dissociation of primase was induced by DNA-dependent binding of PCNA to PolD. Point mutations in the alternative PIP-motif of DP2 abrogated the molecular switching that converts the archaeal replicase from de novo to processive synthesis mode.

Automated summary

This study demonstrated how PolD in the hyperthermophilic archaeon Thermococcus kodakarensis connects primase to the archaeal replisome before interacting with PCNA, with the conserved hydrophobic motif at the C-terminus of the DP2 subunit of PolD playing a critical role in this process. The interaction of PolD with primase is through association with GINS, and cryo-EM analysis revealed a highly flexible PolD-primase complex.