期刊
NUCLEIC ACIDS RESEARCH
卷 49, 期 8, 页码 4599-4612出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkab243
关键词
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资金
- Japan Society for the Promotion of Science (JSPS) [JP20J12260]
- JSPS KAKENHI [JP26242075, JP18K05442, JP18K06089, JP16H01410, JP17H01818]
- JST PRESTO [JPMJPR12L9]
- Platform Project for Supporting Drug Discovery and Life Science Research (Basis of Supporting Innovative Drug Discovery and Life Science Research) from AMED [JP19am0101069, 0673, JP21am0101071, 2054]
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.
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.
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