- EMDB-29351: Cryo-EM structure of S. cerevisiae DNA polymerase alpha-primase c... -
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データベース: EMDB / ID: EMD-29351
タイトル
Cryo-EM structure of S. cerevisiae DNA polymerase alpha-primase complex in the post RNA handoff state
マップデータ
試料
複合体: DNA polymerase alpha/primase complex
タンパク質・ペプチド: DNA primase large subunit
タンパク質・ペプチド: DNA polymerase alpha subunit B
タンパク質・ペプチド: DNA polymerase
タンパク質・ペプチド: DNA primase
DNA: template DNA
RNA: Primer RNA
リガンド: IRON/SULFUR CLUSTER
キーワード
DNA polymerase / primase / DNA replication / DNA BINDING PROTEIN
機能・相同性
機能・相同性情報
alpha DNA polymerase:primase complex / DNA primase activity / lagging strand elongation / DNA replication, synthesis of primer / mitotic DNA replication initiation / leading strand elongation / DNA replication origin binding / DNA replication initiation / 転移酵素; リンを含む基を移すもの; 核酸を移すもの / single-stranded DNA binding ...alpha DNA polymerase:primase complex / DNA primase activity / lagging strand elongation / DNA replication, synthesis of primer / mitotic DNA replication initiation / leading strand elongation / DNA replication origin binding / DNA replication initiation / 転移酵素; リンを含む基を移すもの; 核酸を移すもの / single-stranded DNA binding / 4 iron, 4 sulfur cluster binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / chromatin binding / DNA binding / metal ion binding 類似検索 - 分子機能
DNA polymerase alpha subunit B N-terminal / DNA polymerase alpha, subunit B, N-terminal / DNA polymerase alpha, subunit B / DNA primase, small subunit, eukaryotic/archaeal / DNA primase, large subunit, eukaryotic / DNA primase, small subunit / DNA primase small subunit / DNA primase large subunit, eukaryotic/archaeal / DNA polymerase alpha catalytic subunit, N-terminal domain / DNA polymerase alpha, zinc finger domain superfamily ...DNA polymerase alpha subunit B N-terminal / DNA polymerase alpha, subunit B, N-terminal / DNA polymerase alpha, subunit B / DNA primase, small subunit, eukaryotic/archaeal / DNA primase, large subunit, eukaryotic / DNA primase, small subunit / DNA primase small subunit / DNA primase large subunit, eukaryotic/archaeal / DNA polymerase alpha catalytic subunit, N-terminal domain / DNA polymerase alpha, zinc finger domain superfamily / Eukaryotic and archaeal DNA primase, large subunit / DNA Polymerase alpha zinc finger / DNA polymerase alpha subunit p180 N terminal / Zinc finger, DNA-directed DNA polymerase, family B, alpha / DNA polymerase alpha catalytic subunit, catalytic domain / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / DNA polymerase family B, thumb domain / DNA polymerase family B signature. / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA-directed DNA polymerase, family B, multifunctional domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily 類似検索 - ドメイン・相同性
DNA primase large subunit / DNA polymerase / DNA primase / DNA polymerase alpha subunit B 類似検索 - 構成要素
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM131754
米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM115809
米国
Howard Hughes Medical Institute (HHMI)
M.E.O.
米国
引用
ジャーナル: bioRxiv / 年: 2023 タイトル: Molecular choreography of primer synthesis by the eukaryotic Pol α-primase. 著者: Zuanning Yuan / Roxana Georgescu / Huilin Li / Michael E O'Donnell / 要旨: The eukaryotic polymerase α (Pol α) is a dual-function DNA polymerase/primase complex that synthesizes an RNA-DNA hybrid primer of 20-30 nucleotides for DNA replication. Pol α is composed of Pol1, ...The eukaryotic polymerase α (Pol α) is a dual-function DNA polymerase/primase complex that synthesizes an RNA-DNA hybrid primer of 20-30 nucleotides for DNA replication. Pol α is composed of Pol1, Pol12, Primase 1 (Pri1), and Pri2, with Pol1 and Pri1 containing the DNA polymerase activity and RNA primase activity, respectively, whereas Pol12 and Pri2 serve a structural role. It has been unclear how Pol α hands over an RNA primer made by Pri1 to Pol1 for DNA primer extension, and how the primer length is defined, perhaps due to the difficulty in studying the highly mobile structure. Here we report a comprehensive cryo-EM analysis of the intact 4-subunit yeast Pol α in the apo, primer initiation, primer elongation, RNA primer hand-off from Pri1 to Pol1, and DNA extension states in a 3.5 Å - 5.6 Å resolution range. We found that Pol α is a three-lobed flexible structure. Pri2 functions as a flexible hinge that holds together the catalytic Pol1-core, and the noncatalytic Pol1 CTD that binds to Pol 12 to form a stable platform upon which the other components are organized. In the apo state, Pol1-core is sequestered on the Pol12-Pol1-CTD platform, and Pri1 is mobile perhaps in search of a template. Upon binding a ssDNA template, a large conformation change is induced that enables Pri1 to perform RNA synthesis, and positions Pol1-core to accept the future RNA primed site 50 Å upstream of where Pri1 binds. We reveal in detail the critical point at which Pol1-core takes over the 3'-end of the RNA from Pri1. DNA primer extension appears limited by the spiral motion of Pol1-core while Pri2-CTD stably holds onto the 5' end of the RNA primer. Since both Pri1 and Pol1-core are attached via two linkers to the platform, primer growth will produce stress within this "two-point" attachment that may limit the length of the RNA-DNA hybrid primer. Hence, this study reveals the large and dynamic series of movements that Pol α undergoes to synthesize a primer for DNA replication.