- EMDB-19187: Cryo-EM structure of an MCM helicase single hexamer loaded onto dsDNA. -
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基本情報
登録情報
データベース: EMDB / ID: EMD-19187
タイトル
Cryo-EM structure of an MCM helicase single hexamer loaded onto dsDNA.
マップデータ
Primary map from final 3D refinement in Relion.
試料
複合体: Hexameric MCM helicase complex loaded onto duplex DNA
タンパク質・ペプチド: x 6種
DNA: x 2種
リガンド: x 4種
キーワード
MCM helicase / DNA replication / REPLICATION
機能・相同性
機能・相同性情報
MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / nuclear DNA replication / premeiotic DNA replication / replication fork protection complex / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / Activation of the pre-replicative complex / mitotic DNA replication ...MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / nuclear DNA replication / premeiotic DNA replication / replication fork protection complex / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / Activation of the pre-replicative complex / mitotic DNA replication / CMG complex / nuclear pre-replicative complex / Activation of ATR in response to replication stress / DNA replication preinitiation complex / MCM complex / double-strand break repair via break-induced replication / mitotic DNA replication initiation / single-stranded DNA helicase activity / silent mating-type cassette heterochromatin formation / regulation of DNA-templated DNA replication initiation / DNA strand elongation involved in DNA replication / nuclear replication fork / DNA replication origin binding / DNA replication initiation / subtelomeric heterochromatin formation / DNA helicase activity / transcription elongation by RNA polymerase II / helicase activity / heterochromatin formation / single-stranded DNA binding / DNA helicase / chromosome, telomeric region / DNA replication / DNA damage response / chromatin binding / ATP hydrolysis activity / zinc ion binding / nucleoplasm / ATP binding / nucleus / cytoplasm 類似検索 - 分子機能
ジャーナル: Nat Struct Mol Biol / 年: 2025 タイトル: Cell cycle regulation has shaped replication origins in budding yeast. 著者: Chew Theng Lim / Thomas C R Miller / Kang Wei Tan / Saurabh Talele / Anne Early / Philip East / Humberto Sánchez / Nynke H Dekker / Alessandro Costa / John F X Diffley / 要旨: Eukaryotic DNA replication initiates from genomic loci known as origins. At budding yeast origins like ARS1, a double hexamer (DH) of the MCM replicative helicase is assembled by origin recognition ...Eukaryotic DNA replication initiates from genomic loci known as origins. At budding yeast origins like ARS1, a double hexamer (DH) of the MCM replicative helicase is assembled by origin recognition complex (ORC), Cdc6 and Cdt1 by sequential hexamer loading from two opposed ORC binding sites. Cyclin-dependent kinase (CDK) inhibits DH assembly, which prevents re-replication by restricting helicase loading to the G1 phase. Here, we show that an intrinsically disordered region (IDR) in the Orc2 subunit promotes interaction between ORC and the first loaded, closed-ring MCM hexamer (the MCM-ORC (MO) intermediate). CDK-dependent phosphorylation of this IDR blocks MO formation and DH assembly. We show that MO stabilizes ORC at lower-affinity binding sites required for second hexamer loading. Origins comprising two high-affinity ORC sites can assemble DH efficiently without MO by independently loading single hexamers. Strikingly, these origins escape CDK inhibition in vitro and in vivo. Our work reveals mechanistic plasticity in MCM loading with implications for understanding how CDK regulation has shaped yeast origin evolution and how natural, strong origins might escape cell cycle regulation. We also identify key steps common to loading pathways, with implications for understanding how MCM is loaded in other eukaryotes.
全体 : Hexameric MCM helicase complex loaded onto duplex DNA
全体
名称: Hexameric MCM helicase complex loaded onto duplex DNA
要素
複合体: Hexameric MCM helicase complex loaded onto duplex DNA
タンパク質・ペプチド: DNA replication licensing factor MCM2
タンパク質・ペプチド: DNA replication licensing factor MCM3
タンパク質・ペプチド: DNA replication licensing factor MCM4
タンパク質・ペプチド: Minichromosome maintenance protein 5
タンパク質・ペプチド: DNA replication licensing factor MCM6
タンパク質・ペプチド: DNA replication licensing factor MCM7
DNA: DNA (5'-D(P*GP*CP*AP*TP*GP*CP*AP*TP*GP*CP*AP*TP*GP*CP*AP*TP*GP*CP*AP*T)-3')
DNA: DNA (5'-D(P*AP*TP*GP*CP*AP*TP*GP*CP*AP*TP*GP*CP*AP*TP*GP*CP*AP*T)-3')
リガンド: ADENOSINE-5'-TRIPHOSPHATE
リガンド: MAGNESIUM ION
リガンド: ZINC ION
リガンド: ADENOSINE-5'-DIPHOSPHATE
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超分子 #1: Hexameric MCM helicase complex loaded onto duplex DNA
超分子
名称: Hexameric MCM helicase complex loaded onto duplex DNA タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: #1-#8 詳細: Structural intermediate on path to MCM-double hexamer formation during the process of origin licensing.
モデル: UltrAuFoil R1.2/1.3 / 材質: GOLD / メッシュ: 300 / 支持フィルム - 材質: GRAPHENE OXIDE / 前処理 - タイプ: GLOW DISCHARGE / 前処理 - 時間: 300 sec. / 前処理 - 雰囲気: AIR 詳細: Glow discharge at 40 mA for 5 minutes prior to applying graphene oxide.
凍結
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 283 K / 装置: FEI VITROBOT MARK IV
詳細
Complete biochemical reaction containing the MCM helicase in complex with Cdt1, the helicase loader (the origin recognition complex), the co-factor Cdc6, an origin or replication (DNA) flanked by nucleosomes, the regulatory kinase CDK and an inhibitor of CDK (Sic1). The sample was incubated with ATP yielding a range of structural intermediates in the MCM loading pathway.