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基本情報
登録情報 | データベース: PDB / ID: 7c9a | ||||||||||||
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タイトル | Human RAD51 post-synaptic complexes mutant (V273P, D274G) | ||||||||||||
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![]() | RECOMBINATION / meitotic homologous recombination / DNA repair / ATPase | ||||||||||||
機能・相同性 | ![]() presynaptic intermediate filament cytoskeleton / mitotic recombination-dependent replication fork processing / cellular response to camptothecin / chromosome organization involved in meiotic cell cycle / telomere maintenance via telomere lengthening / positive regulation of DNA ligation / double-strand break repair involved in meiotic recombination / nuclear ubiquitin ligase complex / cellular response to hydroxyurea / replication-born double-strand break repair via sister chromatid exchange ...presynaptic intermediate filament cytoskeleton / mitotic recombination-dependent replication fork processing / cellular response to camptothecin / chromosome organization involved in meiotic cell cycle / telomere maintenance via telomere lengthening / positive regulation of DNA ligation / double-strand break repair involved in meiotic recombination / nuclear ubiquitin ligase complex / cellular response to hydroxyurea / replication-born double-strand break repair via sister chromatid exchange / lateral element / telomere maintenance via recombination / DNA recombinase assembly / regulation of DNA damage checkpoint / mitotic recombination / Impaired BRCA2 binding to PALB2 / DNA strand invasion / DNA strand exchange activity / reciprocal meiotic recombination / Defective homologous recombination repair (HRR) due to BRCA1 loss of function / Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA1 binding function / Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA2/RAD51/RAD51C binding function / Homologous DNA Pairing and Strand Exchange / Resolution of D-loop Structures through Synthesis-Dependent Strand Annealing (SDSA) / Resolution of D-loop Structures through Holliday Junction Intermediates / single-stranded DNA helicase activity / ATP-dependent DNA damage sensor activity / HDR through Single Strand Annealing (SSA) / Impaired BRCA2 binding to RAD51 / regulation of double-strand break repair via homologous recombination / nuclear chromosome / replication fork processing / Transcriptional Regulation by E2F6 / DNA unwinding involved in DNA replication / Presynaptic phase of homologous DNA pairing and strand exchange / ATP-dependent activity, acting on DNA / interstrand cross-link repair / DNA polymerase binding / condensed chromosome / meiotic cell cycle / condensed nuclear chromosome / male germ cell nucleus / cellular response to ionizing radiation / regulation of protein phosphorylation / double-strand break repair via homologous recombination / HDR through Homologous Recombination (HRR) / PML body / Meiotic recombination / site of double-strand break / single-stranded DNA binding / double-stranded DNA binding / DNA recombination / chromosome, telomeric region / mitochondrial matrix / DNA repair / centrosome / DNA damage response / chromatin binding / chromatin / nucleolus / perinuclear region of cytoplasm / enzyme binding / ATP hydrolysis activity / protein-containing complex / mitochondrion / nucleoplasm / ATP binding / identical protein binding / nucleus / cytoplasm / cytosol 類似検索 - 分子機能 | ||||||||||||
生物種 | ![]() | ||||||||||||
手法 | 電子顕微鏡法 / らせん対称体再構成法 / クライオ電子顕微鏡法 / 解像度: 3.43 Å | ||||||||||||
![]() | Chi, H.Y. / Ho, M.C. / Tsai, M.D. / Luo, S.C. / Yeh, H.Y. | ||||||||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Identification of fidelity-governing factors in human recombinases DMC1 and RAD51 from cryo-EM structures. 著者: Shih-Chi Luo / Hsin-Yi Yeh / Wei-Hsuan Lan / Yi-Min Wu / Cheng-Han Yang / Hao-Yen Chang / Guan-Chin Su / Chia-Yi Lee / Wen-Jin Wu / Hung-Wen Li / Meng-Chiao Ho / Peter Chi / Ming-Daw Tsai / ![]() 要旨: Both high-fidelity and mismatch-tolerant recombination, catalyzed by RAD51 and DMC1 recombinases, respectively, are indispensable for genomic integrity. Here, we use cryo-EM, MD simulation and ...Both high-fidelity and mismatch-tolerant recombination, catalyzed by RAD51 and DMC1 recombinases, respectively, are indispensable for genomic integrity. Here, we use cryo-EM, MD simulation and functional analysis to elucidate the structural basis for the mismatch tolerance of DMC1. Structural analysis of DMC1 presynaptic and postsynaptic complexes suggested that the lineage-specific Loop 1 Gln244 (Met243 in RAD51) may help stabilize DNA backbone, whereas Loop 2 Pro274 and Gly275 (Val273/Asp274 in RAD51) may provide an open "triplet gate" for mismatch tolerance. In support, DMC1-Q244M displayed marked increase in DNA dynamics, leading to unobservable DNA map. MD simulation showed highly dispersive mismatched DNA ensemble in RAD51 but well-converged DNA in DMC1 and RAD51-V273P/D274G. Replacing Loop 1 or Loop 2 residues in DMC1 with RAD51 counterparts enhanced DMC1 fidelity, while reciprocal mutations in RAD51 attenuated its fidelity. Our results show that three Loop 1/Loop 2 residues jointly enact contrasting fidelities of DNA recombinases. | ||||||||||||
履歴 |
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構造の表示
ムービー |
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構造ビューア | 分子: ![]() ![]() |
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PDBx/mmCIF形式 | ![]() | 177.2 KB | 表示 | ![]() |
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PDB形式 | ![]() | 140.2 KB | 表示 | ![]() |
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-検証レポート
文書・要旨 | ![]() | 1.1 MB | 表示 | ![]() |
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文書・詳細版 | ![]() | 1.1 MB | 表示 | |
XML形式データ | ![]() | 40 KB | 表示 | |
CIF形式データ | ![]() | 55.8 KB | 表示 | |
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
-関連構造データ
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リンク
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集合体
登録構造単位 | ![]()
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要素
#1: タンパク質 | 分子量: 36949.074 Da / 分子数: 3 / 変異: V273P, D274G / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() ![]() ![]() #2: DNA鎖 | | 分子量: 2692.778 Da / 分子数: 1 / 由来タイプ: 合成 詳細: Authors know the sequence of chains D/E. Chain D: TCA GCT GTT GCC CGT. Chain E: ACG GGC AAC AGC TGA. Since the DNA sequence could not be revealed by the helical reconstruction method, authors ...詳細: Authors know the sequence of chains D/E. Chain D: TCA GCT GTT GCC CGT. Chain E: ACG GGC AAC AGC TGA. Since the DNA sequence could not be revealed by the helical reconstruction method, authors have used poly-T for chain D, poly-A for chain E respectively for model building. 由来: (合成) ![]() #3: DNA鎖 | | 分子量: 2773.904 Da / 分子数: 1 / 由来タイプ: 合成 詳細: Authors know the sequence of chains D/E. Chain D: TCA GCT GTT GCC CGT. Chain E: ACG GGC AAC AGC TGA. Since the DNA sequence could not be revealed by the helical reconstruction method, authors ...詳細: Authors know the sequence of chains D/E. Chain D: TCA GCT GTT GCC CGT. Chain E: ACG GGC AAC AGC TGA. Since the DNA sequence could not be revealed by the helical reconstruction method, authors have used poly-T for chain D, poly-A for chain E respectively for model building. 由来: (合成) ![]() #4: 化合物 | #5: 化合物 | 研究の焦点であるリガンドがあるか | N | |
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-実験情報
-実験
実験 | 手法: 電子顕微鏡法 |
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EM実験 | 試料の集合状態: FILAMENT / 3次元再構成法: らせん対称体再構成法 |
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試料調製
構成要素 | 名称: RAD51-dsDNA filament (V273P D274G mutant) / タイプ: COMPLEX / Entity ID: #1-#3 / 由来: RECOMBINANT |
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由来(天然) | 生物種: ![]() |
由来(組換発現) | 生物種: ![]() ![]() |
緩衝液 | pH: 7.5 詳細: 25 mM Tris-HCl, pH 7.5, 50 mM KCl and 1 mM dithiothreitol) containing 2 mM AMP-PNP and 5 mM CaCl2 |
緩衝液成分 | 濃度: 25 mM / 式: Tris-HCl |
試料 | 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES 詳細: protein sample were applied onto a pre-glow-discharged graphene-oxide coated Quantifoil holey carbon grid (1.2/1.3, 200 mesh) using published protocol |
試料支持 | グリッドの材料: COPPER / グリッドのサイズ: 200 divisions/in. / グリッドのタイプ: Quantifoil R1.2/1.3 |
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: 295 K 詳細: The grids were blotted for 1 sec at 22 degree C with 100% relative humidity and plunge-frozen in liquid ethane cooled by liquid nitrogen using a Vitrobot Mark IV (Thermo Fisher). |
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電子顕微鏡撮影
実験機器 | ![]() モデル: Titan Krios / 画像提供: FEI Company |
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顕微鏡 | モデル: FEI TITAN KRIOS |
電子銃 | 電子線源: ![]() |
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 165000 X / Cs: 2.7 mm / C2レンズ絞り径: 70 µm / アライメント法: COMA FREE |
試料ホルダ | 凍結剤: NITROGEN 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER Residual tilt: 10 mradians |
撮影 | 平均露光時間: 5 sec. / 電子線照射量: 50 e/Å2 / 検出モード: COUNTING フィルム・検出器のモデル: GATAN K2 QUANTUM (4k x 4k) 撮影したグリッド数: 1 |
電子光学装置 | エネルギーフィルター名称: GIF Quantum LS / エネルギーフィルタースリット幅: 30 eV |
画像スキャン | 横: 3838 / 縦: 3710 / 動画フレーム数/画像: 50 / 利用したフレーム数/画像: 1-50 |
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解析
EMソフトウェア |
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CTF補正 | タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||
らせん対称 | 回転角度/サブユニット: 56.18 ° / 軸方向距離/サブユニット: 15.8 Å / らせん対称軸の対称性: C1 | ||||||||||||||||||||||||||||
粒子像の選択 | 選択した粒子像数: 136657 詳細: Filaments were manually picked, and segments were extracted using a box size of 384 pixel and an inter-box distance of ~10% of the box length. | ||||||||||||||||||||||||||||
3次元再構成 | 解像度: 3.43 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 70291 / アルゴリズム: FOURIER SPACE / クラス平均像の数: 4 / 対称性のタイプ: HELICAL |