Open AccessDNA polymerase D temporarily connects primase to the CMG-like helicase before interacting with proliferating cell nuclear antigen
Author(s) -
Keisuke Oki,
Takeshi Yamagami,
Mariko Nagata,
Kouta Mayanagi,
Tomoyuki Shirai,
Naruhiko Adachi,
T. Numata,
Sonoko Ishino,
Yuji Ishino
Publication year - 2021
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/gkab243
Subject(s) - primase , biology , replisome , dna polymerase , dna polymerase ii , dna replication , processivity , dna clamp , helicase , proliferating cell nuclear antigen , microbiology and biotechnology , dna polymerase delta , polymerase , dna , biochemistry , circular bacterial chromosome , rna , reverse transcriptase , gene
The eukaryotic replisome is comprised of three family-B DNA polymerases (Polα, δ and ϵ). Polα forms a stable complex with primase to synthesize short RNA-DNA primers, which are subsequently elongated by Polδ and 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.