- EMDB-13978: S. cerevisiae CMGE nucleating origin DNA melting -
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基本情報
登録情報
データベース: EMDB / ID: EMD-13978
タイトル
S. cerevisiae CMGE nucleating origin DNA melting
マップデータ
PEHNIX Resolve cryoEM density modified map generated from RELION half maps
試料
複合体: S. cerevisiae CMG helicase nucleating origin DNA melting
タンパク質・ペプチド: x 13種
DNA: x 2種
リガンド: x 4種
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 / MCM complex binding / GINS complex ...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 / MCM complex binding / GINS complex / nuclear DNA replication / mitotic DNA replication preinitiation complex assembly / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / SUMO binding / mitotic DNA replication / Activation of the pre-replicative complex / CMG complex / Termination of translesion DNA synthesis / 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 / replication fork protection complex / mitotic DNA replication initiation / mitotic DNA replication checkpoint signaling / mitotic intra-S DNA damage checkpoint signaling / nucleotide-excision repair, DNA gap filling / DNA replication proofreading / single-stranded DNA helicase activity / 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 / silent mating-type cassette heterochromatin formation / mitotic sister chromatid cohesion / DNA strand elongation involved in DNA replication / nuclear chromosome / leading strand elongation / DNA unwinding involved in DNA replication / nuclear replication fork / DNA replication origin binding / Dual incision in TC-NER / DNA replication initiation / subtelomeric heterochromatin formation / error-prone translesion synthesis / DNA helicase activity / base-excision repair, gap-filling / helicase activity / DNA複製 / 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 / mitotic cell cycle / single-stranded DNA binding / 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
DNA polymerase epsilon, subunit B / DNA polymerase epsilon, catalytic subunit A, C-terminal / DNA polymerase epsilon catalytic subunit / Domain of unknown function (DUF1744) / DUF1744 / CDC45 family / DNA replication complex GINS protein Psf2 / CDC45-like protein / GINS complex, subunit Psf1 / GINS complex, subunit Psf3 ...DNA polymerase epsilon, subunit B / DNA polymerase epsilon, catalytic subunit A, C-terminal / DNA polymerase epsilon catalytic subunit / Domain of unknown function (DUF1744) / DUF1744 / CDC45 family / DNA replication complex GINS protein Psf2 / CDC45-like protein / GINS complex, subunit Psf1 / GINS complex, subunit Psf3 / GINS complex, subunit Psf3 superfamily / DNA replication complex GINS protein SLD5, C-terminal / GINS, helical bundle-like domain superfamily / GINS complex protein Sld5, alpha-helical domain / DNA replication complex GINS protein SLD5 C-terminus / GINS complex subunit Sld5 / GINS subunit, domain A / GINS complex protein helical bundle domain / MCM4, winged helix domain / DNA polymerase alpha/delta/epsilon, subunit B / DNA replication licensing factor Mcm5 / DNA polymerase alpha/epsilon subunit B / DNA replication licensing factor Mcm3 / Mini-chromosome maintenance complex protein 4 / DNA replication licensing factor Mcm6 / DNA replication licensing factor Mcm7 / Mcm6, C-terminal winged-helix domain / MCM6 C-terminal winged-helix domain / DNA replication licensing factor Mcm2 / Mini-chromosome maintenance protein 2 / Mini-chromosome maintenance, conserved site / MCM family signature. / MCM N-terminal domain / MCM N-terminal domain / MCM OB domain / MCM OB domain / Mini-chromosome maintenance protein / MCM, AAA-lid domain / MCM P-loop domain / MCM AAA-lid domain / MCM family domain profile. / minichromosome maintenance proteins / MCM domain / DNA polymerase family B, thumb domain / 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 / Winged helix-like DNA-binding domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Nucleic acid-binding, OB-fold / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase 類似検索 - ドメイン・相同性
DNA replication licensing factor MCM5 / DNA replication complex GINS protein PSF2 / DNA replication licensing factor MCM2 / DNA replication complex GINS protein PSF1 / DNA polymerase epsilon catalytic subunit A / DNA replication licensing factor MCM3 / DNA polymerase epsilon subunit B / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM7 / DNA replication licensing factor MCM6 ...DNA replication licensing factor MCM5 / DNA replication complex GINS protein PSF2 / DNA replication licensing factor MCM2 / DNA replication complex GINS protein PSF1 / DNA polymerase epsilon catalytic subunit A / DNA replication licensing factor MCM3 / DNA polymerase epsilon subunit B / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM7 / DNA replication licensing factor MCM6 / DNA replication complex GINS protein SLD5 / Cell division control protein 45 / DNA replication complex GINS protein PSF3 類似検索 - 構成要素
生物種
Saccharomyces cerevisiae (パン酵母) / DNA molecule (デオキシリボ核酸)
H2020 Marie Curie Actions of the European Commission
European Union
European Molecular Biology Organization (EMBO)
1177-2020
European Union
Human Frontier Science Program (HFSP)
LT000834/2020-L
European Union
European Molecular Biology Organization (EMBO)
962-2019
European Union
The Francis Crick Institute
FC001065
European Union
The Francis Crick Institute
FC001066
European Union
Wellcome Trust
106252/Z/14/Z
European Union
European Research Council (ERC)
669424-CHROMOREP
European Union
European Research Council (ERC)
820102
European Union
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.
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.