+データを開く
-基本情報
登録情報 | データベース: EMDB / ID: EMD-14439 | |||||||||||||||||||||||||||||||||
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タイトル | S. cerevisiae CMGE dimer nucleating origin DNA melting | |||||||||||||||||||||||||||||||||
マップデータ | Consensus map of CMGE dimer model generated from EMD EMD-13978 | |||||||||||||||||||||||||||||||||
試料 |
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機能・相同性 | 機能・相同性情報 DNA-templated DNA replication maintenance of fidelity / 遺伝子変換 / Unwinding of DNA / DNA replication initiation / epsilon DNA polymerase complex / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / nuclear DNA replication / MCM complex binding ...DNA-templated DNA replication maintenance of fidelity / 遺伝子変換 / Unwinding of DNA / DNA replication initiation / epsilon DNA polymerase complex / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / nuclear DNA replication / MCM complex binding / GINS complex / mitotic DNA replication preinitiation complex assembly / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / nucleotide-excision repair, DNA gap filling / SUMO binding / mitotic DNA replication / DNA replication proofreading / Activation of the pre-replicative complex / CMG complex / single-stranded DNA 3'-5' DNA exonuclease activity / nuclear pre-replicative complex / Activation of ATR in response to replication stress / MCM complex / DNA replication preinitiation complex / double-strand break repair via break-induced replication / single-stranded DNA helicase activity / mitotic DNA replication checkpoint signaling / replication fork protection complex / mitotic DNA replication initiation / mitotic intra-S DNA damage checkpoint signaling / silent mating-type cassette heterochromatin formation / 加水分解酵素; エステル加水分解酵素; 5'-リン酸モノエステル産生エンドデオキシリボヌクレアーゼ / DNA strand elongation involved in DNA replication / mitotic sister chromatid cohesion / leading strand elongation / nuclear chromosome / DNA unwinding involved in DNA replication / nuclear replication fork / DNA replication origin binding / subtelomeric heterochromatin formation / DNA replication initiation / error-prone translesion synthesis / base-excision repair, gap-filling / DNA helicase activity / DNA複製 / helicase activity / デオキシリボ核酸 / 塩基除去修復 / DNA-templated DNA replication / double-strand break repair via nonhomologous end joining / double-strand break repair / single-stranded DNA binding / mitotic cell cycle / 4 iron, 4 sulfur cluster binding / double-stranded DNA binding / ヘリカーゼ / DNA複製 / chromosome, telomeric region / DNAポリメラーゼ / DNA-directed DNA polymerase activity / hydrolase activity / 細胞周期 / nucleotide binding / mRNA binding / chromatin binding / ATP hydrolysis activity / DNA binding / zinc ion binding / 核質 / ATP binding / metal ion binding / 細胞核 / 細胞質 類似検索 - 分子機能 | |||||||||||||||||||||||||||||||||
生物種 | Saccharomyces cerevisiae (パン酵母) / DNA molecule (デオキシリボ核酸) | |||||||||||||||||||||||||||||||||
手法 | 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 3.4 Å | |||||||||||||||||||||||||||||||||
データ登録者 | Lewis JS / Sousa JS / Costa A | |||||||||||||||||||||||||||||||||
資金援助 | European Union, フランス, 英国, 10件
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引用 | ジャーナル: Nature / 年: 2022 タイトル: Mechanism of replication origin melting nucleated by CMG helicase assembly. 著者: Jacob S Lewis / Marta H Gross / Joana Sousa / Sarah S Henrikus / Julia F Greiwe / Andrea Nans / John F X Diffley / Alessandro Costa / 要旨: The activation of eukaryotic origins of replication occurs in temporally separated steps to ensure that chromosomes are copied only once per cell cycle. First, the MCM helicase is loaded onto duplex ...The activation of eukaryotic origins of replication occurs in temporally separated steps to ensure that chromosomes are copied only once per cell cycle. First, the MCM helicase is loaded onto duplex DNA as an inactive double hexamer. Activation occurs after the recruitment of a set of firing factors that assemble two Cdc45-MCM-GINS (CMG) holo-helicases. CMG formation leads to the underwinding of DNA on the path to the establishment of the replication fork, but whether DNA becomes melted at this stage is unknown. Here we use cryo-electron microscopy to image ATP-dependent CMG assembly on a chromatinized origin, reconstituted in vitro with purified yeast proteins. We find that CMG formation disrupts the double hexamer interface and thereby exposes duplex DNA in between the two CMGs. The two helicases remain tethered, which gives rise to a splayed dimer, with implications for origin activation and replisome integrity. Inside each MCM ring, the double helix becomes untwisted and base pairing is broken. This comes as the result of ATP-triggered conformational changes in MCM that involve DNA stretching and protein-mediated stabilization of three orphan bases. Mcm2 pore-loop residues that engage DNA in our structure are dispensable for double hexamer loading and CMG formation, but are essential to untwist the DNA and promote replication. Our results explain how ATP binding nucleates origin DNA melting by the CMG and maintains replisome stability at initiation. | |||||||||||||||||||||||||||||||||
履歴 |
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-構造の表示
添付画像 |
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-ダウンロードとリンク
-EMDBアーカイブ
マップデータ | emd_14439.map.gz | 43.5 MB | EMDBマップデータ形式 | |
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ヘッダ (付随情報) | emd-14439-v30.xml emd-14439.xml | 39.3 KB 39.3 KB | 表示 表示 | EMDBヘッダ |
画像 | emd_14439.png | 83.3 KB | ||
アーカイブディレクトリ | http://ftp.pdbj.org/pub/emdb/structures/EMD-14439 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-14439 | HTTPS FTP |
-関連構造データ
-リンク
EMDBのページ | EMDB (EBI/PDBe) / EMDataResource |
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「今月の分子」の関連する項目 |
-マップ
ファイル | ダウンロード / ファイル: emd_14439.map.gz / 形式: CCP4 / 大きさ: 60.2 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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注釈 | Consensus map of CMGE dimer model generated from EMD EMD-13978 | ||||||||||||||||||||||||||||||||||||
投影像・断面図 | 画像のコントロール
画像は Spider により作成 これらの図は立方格子座標系で作成されたものです | ||||||||||||||||||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 1.08 Å | ||||||||||||||||||||||||||||||||||||
密度 |
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対称性 | 空間群: 1 | ||||||||||||||||||||||||||||||||||||
詳細 | EMDB XML:
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-添付データ
-試料の構成要素
+全体 : S. cerevisiae CMGE dimer nucleating origin DNA melting
+超分子 #1: S. cerevisiae CMGE dimer nucleating origin DNA melting
+分子 #1: DNA replication licensing factor MCM2
+分子 #2: DNA replication licensing factor MCM3
+分子 #3: DNA replication licensing factor MCM4
+分子 #4: DNA helicase
+分子 #5: DNA replication licensing factor MCM6
+分子 #6: DNA replication licensing factor MCM7
+分子 #9: DNA replication complex GINS protein PSF3
+分子 #10: DNA replication complex GINS protein SLD5
+分子 #11: Cell division control protein 45
+分子 #12: DNA polymerase epsilon subunit B
+分子 #13: DNA replication complex GINS protein PSF1
+分子 #14: DNA replication complex GINS protein PSF2
+分子 #15: DNA polymerase epsilon catalytic subunit A
+分子 #7: DNA (53-MER)
+分子 #8: DNA (53-MER)
+分子 #16: ADENOSINE-5'-TRIPHOSPHATE
+分子 #17: ZINC ION
+分子 #18: MAGNESIUM ION
+分子 #19: ADENOSINE-5'-DIPHOSPHATE
-実験情報
-構造解析
手法 | クライオ電子顕微鏡法 |
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解析 | 単粒子再構成法 |
試料の集合状態 | particle |
-試料調製
緩衝液 | pH: 7.5 |
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凍結 | 凍結剤: ETHANE / 装置: FEI VITROBOT MARK IV |
詳細 | four microlitres of sample was applied on a grid and incubated for 2 min at room temperature before blotting with filter paper for 5.5 s and plunge-freezing in liquid ethane. |
-電子顕微鏡法
顕微鏡 | FEI TITAN KRIOS |
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電子線 | 加速電圧: 300 kV / 電子線源: FIELD EMISSION GUN |
電子光学系 | C2レンズ絞り径: 50.0 µm / 照射モード: OTHER / 撮影モード: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / 最大 デフォーカス(公称値): 4.4 µm / 最小 デフォーカス(公称値): 2.0 µm / 倍率(公称値): 130000 |
試料ステージ | 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER ホルダー冷却材: NITROGEN |
撮影 | フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 検出モード: COUNTING / 撮影したグリッド数: 2 / 実像数: 65286 / 平均露光時間: 10.0 sec. / 平均電子線量: 1.6 e/Å2 |
実験機器 | モデル: Titan Krios / 画像提供: FEI Company |
-画像解析
初期 角度割当 | タイプ: MAXIMUM LIKELIHOOD / ソフトウェア - 名称: RELION (ver. 3.1) |
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最終 角度割当 | タイプ: MAXIMUM LIKELIHOOD / ソフトウェア - 名称: RELION (ver. 3.1) |
最終 再構成 | 解像度のタイプ: BY AUTHOR / 解像度: 3.4 Å / 解像度の算出法: FSC 0.143 CUT-OFF / ソフトウェア - 名称: RELION (ver. 3.1) / 使用した粒子像数: 71348 |