+データを開く
-基本情報
登録情報 | データベース: PDB / ID: 8un0 | ||||||
---|---|---|---|---|---|---|---|
タイトル | Atomic model of the human CTF18-RFC-PCNA-DNA ternary complex with cracked and closed PCNA (state 7) | ||||||
要素 |
| ||||||
キーワード | REPLICATION/DNA / DNA clamp loader complex / REPLICATION-DNA complex | ||||||
機能・相同性 | 機能・相同性情報 positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / PCNA-p21 complex / Elg1 RFC-like complex / DNA replication factor C complex / Ctf18 RFC-like complex / response to organophosphorus / mitotic telomere maintenance via semi-conservative replication / DNA clamp loader activity / purine-specific mismatch base pair DNA N-glycosylase activity ...positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / PCNA-p21 complex / Elg1 RFC-like complex / DNA replication factor C complex / Ctf18 RFC-like complex / response to organophosphorus / mitotic telomere maintenance via semi-conservative replication / DNA clamp loader activity / purine-specific mismatch base pair DNA N-glycosylase activity / positive regulation of DNA-directed DNA polymerase activity / 核ラミナ / MutLalpha complex binding / Polymerase switching / Telomere C-strand (Lagging Strand) Synthesis / Processive synthesis on the lagging strand / PCNA complex / Removal of the Flap Intermediate / Processive synthesis on the C-strand of the telomere / Mismatch repair (MMR) directed by MSH2:MSH3 (MutSbeta) / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Transcription of E2F targets under negative control by DREAM complex / Polymerase switching on the C-strand of the telomere / Removal of the Flap Intermediate from the C-strand / replisome / DNA strand elongation involved in DNA replication / response to L-glutamate / DNA duplex unwinding / HDR through Single Strand Annealing (SSA) / histone acetyltransferase binding / Impaired BRCA2 binding to RAD51 / leading strand elongation / DNA synthesis involved in DNA repair / DNA polymerase processivity factor activity / G1/S-Specific Transcription / replication fork processing / response to dexamethasone / nuclear replication fork / Presynaptic phase of homologous DNA pairing and strand exchange / SUMOylation of DNA replication proteins / estrous cycle / PCNA-Dependent Long Patch Base Excision Repair / cyclin-dependent protein kinase holoenzyme complex / DNAミスマッチ修復 / DNA修復 / ATP-dependent activity, acting on DNA / response to cadmium ion / Activation of ATR in response to replication stress / DNA polymerase binding / base-excision repair, gap-filling / epithelial cell differentiation / positive regulation of DNA repair / Translesion synthesis by REV1 / Translesion synthesis by POLK / Gap-filling DNA repair synthesis and ligation in GG-NER / Translesion synthesis by POLI / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / DNA複製 / positive regulation of DNA replication / male germ cell nucleus / liver regeneration / nuclear estrogen receptor binding / Recognition of DNA damage by PCNA-containing replication complex / Termination of translesion DNA synthesis / Translesion Synthesis by POLH / HDR through Homologous Recombination (HRR) / G2/M DNA damage checkpoint / Dual Incision in GG-NER / DNA-templated DNA replication / receptor tyrosine kinase binding / cellular response to hydrogen peroxide / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / cellular response to UV / cellular response to xenobiotic stimulus / response to estradiol / E3 ubiquitin ligases ubiquitinate target proteins / heart development / Processing of DNA double-strand break ends / Regulation of TP53 Activity through Phosphorylation / DNA複製 / chromosome, telomeric region / damaged DNA binding / nuclear body / 細胞周期 / DNA修復 / 中心体 / chromatin binding / クロマチン / protein-containing complex binding / negative regulation of transcription by RNA polymerase II / enzyme binding / ATP hydrolysis activity / DNA binding / extracellular exosome / 核質 / ATP binding / 生体膜 / identical protein binding / 細胞核 類似検索 - 分子機能 | ||||||
生物種 | Homo sapiens (ヒト) 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: 3 Å | ||||||
データ登録者 | Wang, F. / He, Q. / Li, H. | ||||||
資金援助 | 米国, 1件
| ||||||
引用 | ジャーナル: Proc Natl Acad Sci U S A / 年: 2024 タイトル: Cryo-EM reveals a nearly complete PCNA loading process and unique features of the human alternative clamp loader CTF18-RFC. 著者: Qing He / Feng Wang / Michael E O'Donnell / Huilin Li / 要旨: The DNA sliding clamp PCNA is a multipurpose platform for DNA polymerases and many other proteins involved in DNA metabolism. The topologically closed PCNA ring needs to be cracked open and loaded ...The DNA sliding clamp PCNA is a multipurpose platform for DNA polymerases and many other proteins involved in DNA metabolism. The topologically closed PCNA ring needs to be cracked open and loaded onto DNA by a clamp loader, e.g., the well-studied pentameric ATPase complex RFC (RFC1-5). The CTF18-RFC complex is an alternative clamp loader found recently to bind the leading strand DNA polymerase ε and load PCNA onto leading strand DNA, but its structure and the loading mechanism have been unknown. By cryo-EM analysis of in vitro assembled human CTF18-RFC-DNA-PCNA complex, we have captured seven loading intermediates, revealing a detailed PCNA loading mechanism onto a 3'-ss/dsDNA junction by CTF18-RFC. Interestingly, the alternative loader has evolved a highly mobile CTF18 AAA+ module likely to lower the loading activity, perhaps to avoid competition with the RFC and to limit its role to leading strand clamp loading. To compensate for the lost stability due to the mobile AAA+ module, CTF18 has evolved a unique β-hairpin motif that reaches across RFC2 to interact with RFC5, thereby stabilizing the pentameric complex. Further, we found that CTF18 also contains a separation pin to locally melt DNA from the 3'-end of the primer; this ensures its ability to load PCNA to any 3'-ss/dsDNA junction, facilitated by the binding energy of the E-plug to the major groove. Our study reveals unique structural features of the human CTF18-RFC and contributes to a broader understanding of PCNA loading by the alternative clamp loaders. | ||||||
履歴 |
|
-構造の表示
構造ビューア | 分子: MolmilJmol/JSmol |
---|
-ダウンロードとリンク
-ダウンロード
PDBx/mmCIF形式 | 8un0.cif.gz | 560.2 KB | 表示 | PDBx/mmCIF形式 |
---|---|---|---|---|
PDB形式 | pdb8un0.ent.gz | 442.3 KB | 表示 | PDB形式 |
PDBx/mmJSON形式 | 8un0.json.gz | ツリー表示 | PDBx/mmJSON形式 | |
その他 | その他のダウンロード |
-検証レポート
アーカイブディレクトリ | https://data.pdbj.org/pub/pdb/validation_reports/un/8un0 ftp://data.pdbj.org/pub/pdb/validation_reports/un/8un0 | HTTPS FTP |
---|
-関連構造データ
関連構造データ | 42389MC 8umtC 8umuC 8umvC 8umwC 8umyC 8unjC 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 (文献) |
---|---|
類似構造データ | 類似検索 - 機能・相同性F&H 検索 |
-リンク
-集合体
登録構造単位 |
|
---|---|
1 |
|
-要素
-タンパク質 , 2種, 4分子 AFGH
#1: タンパク質 | 分子量: 107523.984 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: CHTF18 発現宿主: Insect cell expression vector pTIE1 (その他) 参照: UniProt: Q8WVB6 |
---|---|
#6: タンパク質 | 分子量: 28795.752 Da / 分子数: 3 / 由来タイプ: 組換発現 / 由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: PCNA 発現宿主: Insect cell expression vector pTIE1 (その他) 参照: UniProt: P12004 |
-Replication factor C subunit ... , 4種, 4分子 BCDE
#2: タンパク質 | 分子量: 39203.207 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: RFC2 発現宿主: Insect cell expression vector pTIE1 (その他) 参照: UniProt: P35250 |
---|---|
#3: タンパク質 | 分子量: 38545.512 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: RFC5 発現宿主: Insect cell expression vector pTIE1 (その他) 参照: UniProt: P40937 |
#4: タンパク質 | 分子量: 39735.711 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: RFC4 発現宿主: Insect cell expression vector pTIE1 (その他) 参照: UniProt: P35249 |
#5: タンパク質 | 分子量: 40614.332 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: RFC3 発現宿主: Insect cell expression vector pTIE1 (その他) 参照: UniProt: P40938 |
-DNA鎖 , 2種, 2分子 IJ
#7: DNA鎖 | 分子量: 12214.818 Da / 分子数: 1 / 由来タイプ: 合成 / 由来: (合成) synthetic construct (人工物) |
---|---|
#8: DNA鎖 | 分子量: 6136.008 Da / 分子数: 1 / 由来タイプ: 合成 / 由来: (合成) synthetic construct (人工物) |
-非ポリマー , 3種, 9分子
#9: 化合物 | ChemComp-MG / #10: 化合物 | ChemComp-AGS / #11: 化合物 | ChemComp-ADP / | |
---|
-詳細
研究の焦点であるリガンドがあるか | Y |
---|
-実験情報
-実験
実験 | 手法: 電子顕微鏡法 |
---|---|
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 |
-試料調製
構成要素 | 名称: the human clamp-clamp loader complex PCNA-CTF18 / タイプ: COMPLEX / Entity ID: #1-#8 / 由来: RECOMBINANT |
---|---|
分子量 | 値: 0.3 MDa / 実験値: YES |
由来(天然) | 生物種: Homo sapiens (ヒト) |
由来(組換発現) | 生物種: Insect cell expression vector pTIE1 (その他) |
緩衝液 | pH: 7.5 |
試料 | 濃度: 0.3 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES |
急速凍結 | 装置: FEI VITROBOT MARK IV / 凍結剤: ETHANE / 湿度: 100 % / 凍結前の試料温度: 280 K |
-電子顕微鏡撮影
実験機器 | モデル: Titan Krios / 画像提供: FEI Company |
---|---|
顕微鏡 | モデル: FEI TITAN KRIOS |
電子銃 | 電子線源: FIELD EMISSION GUN / 加速電圧: 300 kV / 照射モード: FLOOD BEAM |
電子レンズ | モード: BRIGHT FIELDBright-field microscopy / 最大 デフォーカス(公称値): 1900 nm / 最小 デフォーカス(公称値): 1200 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 (6k x 4k) |
-解析
CTF補正 | タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION |
---|---|
3次元再構成 | 解像度: 3 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 75789 / 対称性のタイプ: POINT |
原子モデル構築 | B value: 123 / プロトコル: RIGID BODY FIT / 空間: REAL |