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

Keisuke Oki, Takeshi Yamagami, Mariko Nagata, Kouta Mayanagi, Tsuyoshi Shirai, Naruhiko Adachi, Tomoyuki Numata, Sonoko Ishino, Yoshizumi Ishino

Research output: Contribution to journalArticlepeer-review

Abstract

The eukaryotic replisome is comprised of three family-B DNA polymerases (Polα, δand ε). Polα forms a stable complex with primase to synthesize short RNA-DNA primers, which are subsequently elongated by Polδand Polε 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 how it 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.

Original languageEnglish
Pages (from-to)4599-4612
Number of pages14
JournalNucleic acids research
Volume49
Issue number8
DOIs
Publication statusPublished - May 7 2021

All Science Journal Classification (ASJC) codes

  • Genetics

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