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基本情報
登録情報 | データベース: PDB / ID: 8foj | ||||||||||||
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タイトル | Cryo-EM structure of S. cerevisiae DNA polymerase alpha-primase complex in the post RNA handoff state | ||||||||||||
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![]() | DNA BINDING PROTEIN / DNA polymerase / primase / DNA replication | ||||||||||||
機能・相同性 | ![]() 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 / chromatin binding / nucleotide binding / DNA binding / metal ion binding 類似検索 - 分子機能 | ||||||||||||
生物種 | ![]() ![]() synthetic construct (人工物) | ||||||||||||
手法 | 電子顕微鏡法 / 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 4.8 Å | ||||||||||||
![]() | Yuan, Z. / Georgescu, R. / Li, H. / O'Donnell, M. | ||||||||||||
資金援助 | ![]()
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![]() | ![]() 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) synthesizes an RNA-DNA hybrid primer of 20-30 nucleotides. Pol α is composed of Pol1, Pol12, Primase 1 (Pri1), and Pri2. Pol1 and Pri1 contain the DNA ...The eukaryotic polymerase α (Pol α) synthesizes an RNA-DNA hybrid primer of 20-30 nucleotides. Pol α is composed of Pol1, Pol12, Primase 1 (Pri1), and Pri2. Pol1 and Pri1 contain the DNA polymerase and RNA primase activities, respectively. It has been unclear how Pol α hands over an RNA primer from Pri1 to Pol1 for DNA primer extension, and how the primer length is defined. Here we report the cryo-EM analysis of yeast Pol α in the apo, primer initiation, primer elongation, RNA primer hand-off from Pri1 to Pol1, and DNA extension states, revealing a series of very large movements. We reveal a critical point at which Pol1-core moves to take over the 3'-end of the RNA from Pri1. DNA extension is limited by a spiral motion of Pol1-core. Since both Pri1 and Pol1-core are flexibly attached to a stable platform, primer growth produces stress that limits the primer length. #1: 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. | ||||||||||||
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構造の表示
構造ビューア | 分子: ![]() ![]() |
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PDBx/mmCIF形式 | ![]() | 426.7 KB | 表示 | ![]() |
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PDB形式 | ![]() | 324.1 KB | 表示 | ![]() |
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-検証レポート
文書・要旨 | ![]() | 1 MB | 表示 | ![]() |
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文書・詳細版 | ![]() | 1.1 MB | 表示 | |
XML形式データ | ![]() | 66 KB | 表示 | |
CIF形式データ | ![]() | 99.7 KB | 表示 | |
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
-関連構造データ
関連構造データ | ![]() 29351MC ![]() 8focC ![]() 8fodC ![]() 8foeC ![]() 8fohC ![]() 8fokC M: このデータのモデリングに利用したマップデータ C: 同じ文献を引用 ( |
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類似構造データ | 類似検索 - 機能・相同性 ![]() |
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リンク
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集合体
登録構造単位 | ![]()
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1 | ![]()
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要素
-タンパク質 , 4種, 4分子 1ABC
#1: タンパク質 | 分子量: 167027.766 Da / 分子数: 1 / 由来タイプ: 組換発現 由来: (組換発現) ![]() ![]() 遺伝子: POL1 / 発現宿主: ![]() ![]() |
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#2: タンパク質 | 分子量: 47760.367 Da / 分子数: 1 / 由来タイプ: 組換発現 由来: (組換発現) ![]() ![]() 遺伝子: PRI1 / 発現宿主: ![]() ![]() |
#3: タンパク質 | 分子量: 62348.551 Da / 分子数: 1 / 由来タイプ: 組換発現 由来: (組換発現) ![]() ![]() 遺伝子: PRI2, GI527_G0003596 / 発現宿主: ![]() ![]() |
#4: タンパク質 | 分子量: 78865.938 Da / 分子数: 1 / 由来タイプ: 組換発現 由来: (組換発現) ![]() ![]() 遺伝子: POL12, GI527_G0000165 / 発現宿主: ![]() ![]() |
-DNA鎖 / RNA鎖 / 非ポリマー , 3種, 3分子 TP![](data/chem/img/SF4.gif)
![](data/chem/img/SF4.gif)
#5: DNA鎖 | 分子量: 3286.136 Da / 分子数: 1 / 由来タイプ: 合成 / 由来: (合成) synthetic construct (人工物) |
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#6: RNA鎖 | 分子量: 3279.052 Da / 分子数: 1 / 由来タイプ: 合成 / 由来: (合成) synthetic construct (人工物) |
#7: 化合物 | ChemComp-SF4 / |
-詳細
研究の焦点であるリガンドがあるか | N |
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-実験情報
-実験
実験 | 手法: 電子顕微鏡法 |
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EM実験 | 試料の集合状態: PARTICLE / 3次元再構成法: 単粒子再構成法 |
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試料調製
構成要素 | 名称: DNA polymerase alpha/primase complex / タイプ: COMPLEX / Entity ID: #3-#4, #1-#2, #5-#6 / 由来: RECOMBINANT |
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由来(天然) | 生物種: ![]() ![]() |
由来(組換発現) | 生物種: ![]() ![]() |
緩衝液 | pH: 7.4 |
試料 | 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES |
急速凍結 | 凍結剤: ETHANE |
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電子顕微鏡撮影
実験機器 | ![]() モデル: Titan Krios / 画像提供: FEI Company |
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顕微鏡 | モデル: FEI TITAN KRIOS |
電子銃 | 電子線源: ![]() |
電子レンズ | モード: BRIGHT FIELD / 最大 デフォーカス(公称値): 2500 nm / 最小 デフォーカス(公称値): 1500 nm |
撮影 | 電子線照射量: 60 e/Å2 フィルム・検出器のモデル: GATAN K3 BIOQUANTUM (6k x 4k) |
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解析
CTF補正 | タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION |
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3次元再構成 | 解像度: 4.8 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 650973 / 対称性のタイプ: POINT |