EMDB-15309: Pol alpha - replisome complex containing Ctf4. Engaged on a DNA f...

基本情報

登録情報

データベース: EMDB / ID: EMD-15309

• タイトル: Pol alpha - replisome complex containing Ctf4. Engaged on a DNA fork containing a 60 nucleotide lagging strand.
• マップデータ: Pol alpha - replisome complex containing Ctf4. Unsharpened.

試料

• 複合体: Replisome - pol alpha complex

• キーワード: Replication / helicase / polymerase / pol alpha / priming

機能・相同性

分子機能:

Inhibition of replication initiation of damaged DNA by RB1/E2F1 / establishment of sister chromatid cohesion / H3-H4 histone complex chaperone activity / maintenance of DNA repeat elements / Unwinding of DNA / replication fork arrest / Cul8-RING ubiquitin ligase complex / regulation of nuclear cell cycle DNA replication / DNA replication initiation / meiotic chromosome segregation / RNA-templated DNA biosynthetic process / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / DNA strand elongation involved in mitotic DNA replication / MCM complex binding / Processive synthesis on the lagging strand / GINS complex / nuclear DNA replication / mitotic DNA replication preinitiation complex assembly / Removal of the Flap Intermediate / premeiotic DNA replication / Polymerase switching / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / telomere capping / anaphase-promoting complex binding / mitotic DNA replication / alpha DNA polymerase:primase complex / Activation of the pre-replicative complex / CMG complex / nuclear pre-replicative complex / DNA replication checkpoint signaling / DNA primase activity / establishment of mitotic sister chromatid cohesion / Activation of ATR in response to replication stress / DNA replication preinitiation complex / MCM complex / replication fork protection complex / primosome complex / DNA replication, synthesis of primer / mitotic DNA replication checkpoint signaling / lagging strand elongation / double-strand break repair via break-induced replication / cellular response to osmotic stress / mitotic DNA replication initiation / single-stranded DNA helicase activity / regulation of DNA-templated DNA replication initiation / mitotic intra-S DNA damage checkpoint signaling / DNA strand elongation involved in DNA replication / silent mating-type cassette heterochromatin formation / mitotic sister chromatid cohesion / nuclear chromosome / DNA synthesis involved in DNA repair / DNA biosynthetic process / leading strand elongation / DNA unwinding involved in DNA replication / replication fork processing / nuclear replication fork / mitotic G2 DNA damage checkpoint signaling / DNA replication origin binding / DNA replication initiation / subtelomeric heterochromatin formation / Ub-specific processing proteases / DNA helicase activity / telomere maintenance / nuclear periphery / replication fork / meiotic cell cycle / helicase activity / transcription elongation by RNA polymerase II / heterochromatin formation / DNA-templated DNA replication / 転移酵素; リンを含む基を移すもの; 核酸を移すもの / double-strand break repair / nucleosome assembly / nuclear envelope / mitotic cell cycle / single-stranded DNA binding / 4 iron, 4 sulfur cluster binding / DNA helicase / DNA replication / DNA-directed DNA polymerase / chromosome, telomeric region / DNA-directed DNA polymerase activity / protein stabilization / DNA repair / nucleotide binding / DNA damage response / chromatin binding / ATP hydrolysis activity / mitochondrion / DNA binding / nucleoplasm / ATP binding / identical protein binding / nucleus / metal ion binding / cytosol / cytoplasm

ドメイン・相同性:

DNA replication checkpoint mediator, MRC1 domain / MRC1-like domain / Claspin / Chromosome segregation in meiosis protein 3 / TIPIN/Csm3/Swi3 / Replication Fork Protection Component Swi3 / Timeless / : / DNA polymerase alpha subunit B, OB domain / Timeless, N-terminal / Timeless protein / : / DNA polymerase alpha-binding protein Ctf4, C-terminal domain / DNA polymerase alpha, subunit B / Minichromosome loss protein Mcl1, middle region / Minichromosome loss protein, Mcl1, middle region / 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 / : / MCM3 winged helix domain / GINS/PriA/YqbF domain / 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 polymerase alpha/epsilon subunit B / : / MCM5, C-terminal domain / DNA replication licensing factor Mcm5 / 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 / Mini-chromosome maintenance protein / MCM, AAA-lid domain / MCM P-loop domain / MCM OB domain / MCM AAA-lid domain / MCM family domain profile. / minichromosome maintenance proteins / MCM domain / DNA polymerase family B, thumb domain / DNA-directed DNA polymerase, family B, multifunctional 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 polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Ribonuclease H superfamily / Ribonuclease H-like superfamily / WD40-repeat-containing domain superfamily / Winged helix-like DNA-binding domain superfamily / WD40/YVTN repeat-like-containing 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

構成要素:

DNA primase small subunit / DNA polymerase alpha catalytic subunit A / DNA primase large subunit / DNA replication licensing factor MCM3 / Mediator of replication checkpoint protein 1 / DNA replication licensing factor MCM2 / Minichromosome maintenance protein 5 / DNA replication licensing factor MCM4 / DNA polymerase alpha subunit B / DNA replication licensing factor MCM7 / DNA replication complex GINS protein PSF2 / DNA replication licensing factor MCM6 / Topoisomerase 1-associated factor 1 / DNA polymerase alpha-binding protein / DNA replication complex GINS protein SLD5 / Chromosome segregation in meiosis protein 3 / Cell division control protein 45 / DNA replication complex GINS protein PSF3 / DNA replication complex GINS protein PSF1

• 生物種: Saccharomyces cerevisiae (パン酵母)
• 手法: 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 4.64 Å
• データ登録者: Jones ML / Yeeles JTP

資金援助

英国, 1件

Organization	Grant number	国
Medical Research Council (MRC, United Kingdom)	MC_UP_1201/12	英国

• 引用: ジャーナル: Mol Cell / 年: 2023; タイトル: How Pol α-primase is targeted to replisomes to prime eukaryotic DNA replication.; 著者: Morgan L Jones / Valentina Aria / Yasemin Baris / Joseph T P Yeeles / ; 要旨: During eukaryotic DNA replication, Pol α-primase generates primers at replication origins to start leading-strand synthesis and every few hundred nucleotides during discontinuous lagging-strand replication. How Pol α-primase is targeted to replication forks to prime DNA synthesis is not fully understood. Here, by determining cryoelectron microscopy (cryo-EM) structures of budding yeast and human replisomes containing Pol α-primase, we reveal a conserved mechanism for the coordination of priming by the replisome. Pol α-primase binds directly to the leading edge of the CMG (CDC45-MCM-GINS) replicative helicase via a complex interaction network. The non-catalytic PRIM2/Pri2 subunit forms two interfaces with CMG that are critical for in vitro DNA replication and yeast cell growth. These interactions position the primase catalytic subunit PRIM1/Pri1 directly above the exit channel for lagging-strand template single-stranded DNA (ssDNA), revealing why priming occurs efficiently only on the lagging-strand template and elucidating a mechanism for Pol α-primase to overcome competition from RPA to initiate primer synthesis.

履歴

登録	2022年7月4日	-
ヘッダ(付随情報) 公開	2023年8月2日	-
マップ公開	2023年8月2日	-
更新	2023年8月30日	-
現状	2023年8月30日	処理サイト: PDBe / 状態: 公開

マップ

• ファイル: ダウンロード / ファイル: emd_15309.map.gz / 形式: CCP4 / 大きさ: 40.6 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• 注釈: Pol alpha - replisome complex containing Ctf4. Unsharpened.
• ボクセルのサイズ: X=Y=Z: 2.26727 Å

密度

表面レベル	登録者による: 1.43
最小 - 最大	-3.500969 - 8.684388
平均 (標準偏差)	0.0049348124 (±0.36013365)

• 対称性: 空間群: 1

詳細

EMDB XML:

マップ形状	Axis order X Y Z Origin 0 0 0 サイズ 220 220 220 Spacing 220 220 220
セル	A=B=C: 498.8 Å α=β=γ: 90.0 °

添付データ

追加マップ: Locally filtered map

• ファイル: emd_15309_additional_1.map
• 注釈: Locally filtered map

ハーフマップ: Half map

• ファイル: emd_15309_half_map_1.map
• 注釈: Half map

ハーフマップ: Half map

• ファイル: emd_15309_half_map_2.map
• 注釈: Half map

試料の構成要素

全体 : Replisome - pol alpha complex

• 全体: 名称: Replisome - pol alpha complex

要素

• 複合体: Replisome - pol alpha complex

超分子 #1: Replisome - pol alpha complex

• 超分子: 名称: Replisome - pol alpha complex / タイプ: complex / ID: 1 / 親要素: 0 ; 詳細: S. cerevisiae pol alpha bound to the core replisome engaged with a fork DNA substrate containing a 60 nucleotide lagging strand.
• 由来(天然): 生物種: Saccharomyces cerevisiae (パン酵母)

実験情報

構造解析

• 手法: クライオ電子顕微鏡法
• 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: 7.6
• 凍結: 凍結剤: ETHANE

電子顕微鏡法

• 顕微鏡: FEI TITAN KRIOS
• 撮影: フィルム・検出器のモデル: GATAN K3 (6k x 4k) / 平均電子線量: 40.184 e/Ų
• 電子線: 加速電圧: 300 kV / 電子線源: FIELD EMISSION GUN
• 電子光学系: 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / 最大 デフォーカス（公称値）: 3.5 µm / 最小 デフォーカス（公称値）: 1.5 µm
• 実験機器: モデル: Titan Krios / 画像提供: FEI Company

画像解析

• 初期モデル: モデルのタイプ: OTHER
• 最終 再構成: 解像度のタイプ: BY AUTHOR / 解像度: 4.64 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 使用した粒子像数: 24127
• 初期 角度割当: タイプ: ANGULAR RECONSTITUTION
• 最終 角度割当: タイプ: ANGULAR RECONSTITUTION