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- PDB-7c9c: Human DMC1 pre-synaptic complexes -

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Basic information

Entry
Database: PDB / ID: 7c9c
TitleHuman DMC1 pre-synaptic complexes
Components
  • DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
  • Meiotic recombination protein DMC1/LIM15 homologGenetic recombination
KeywordsRECOMBINATION/DNA / meiotic homologous recombination / DNA repair / ATPase / Recombination / RECOMBINATION-DNA complex
Function / homology
Function and homology information


female gamete generation / chromosome organization involved in meiotic cell cycle / DNA recombinase assembly / homologous chromosome pairing at meiosis / double-strand break repair involved in meiotic recombination / mitotic recombination / DNA strand invasion / DNA strand exchange activity / lateral element / oocyte maturation ...female gamete generation / chromosome organization involved in meiotic cell cycle / DNA recombinase assembly / homologous chromosome pairing at meiosis / double-strand break repair involved in meiotic recombination / mitotic recombination / DNA strand invasion / DNA strand exchange activity / lateral element / oocyte maturation / reciprocal meiotic recombination / ATP-dependent DNA damage sensor activity / male meiosis I / spermatid development / ATP-dependent activity, acting on DNA / ovarian follicle development / meiotic cell cycle / condensed nuclear chromosome / Meiotic recombination / single-stranded DNA binding / site of double-strand break / chromosome / double-stranded DNA binding / spermatogenesis / chromosome, telomeric region / ATP hydrolysis activity / DNA binding / ATP binding / identical protein binding / nucleus
Similarity search - Function
Meiotic recombination protein Dmc1 / DNA recombination and repair protein, RecA-like / DNA recombination and repair protein Rad51-like, C-terminal / Rad51 / DNA recombination and repair protein RecA, monomer-monomer interface / RecA family profile 2. / DNA recombination and repair protein RecA-like, ATP-binding domain / RecA family profile 1. / DNA repair Rad51/transcription factor NusA, alpha-helical / Helix-hairpin-helix domain ...Meiotic recombination protein Dmc1 / DNA recombination and repair protein, RecA-like / DNA recombination and repair protein Rad51-like, C-terminal / Rad51 / DNA recombination and repair protein RecA, monomer-monomer interface / RecA family profile 2. / DNA recombination and repair protein RecA-like, ATP-binding domain / RecA family profile 1. / DNA repair Rad51/transcription factor NusA, alpha-helical / Helix-hairpin-helix domain / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / DNA / Meiotic recombination protein DMC1/LIM15 homolog
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.33 Å
AuthorsLuo, S.C. / Yeh, H.Y. / Chi, P. / Ho, M.C. / Tsai, M.D.
Funding support Taiwan, 3items
OrganizationGrant numberCountry
Academia Sinica (Taiwan)AS-KPQ-109-TPP2 Taiwan
Academia Sinica (Taiwan)AS-CFII-108-110 Taiwan
Academia Sinica (Taiwan)AS-KPQ-105-TPP Taiwan
CitationJournal: Nat Commun / Year: 2021
Title: Identification of fidelity-governing factors in human recombinases DMC1 and RAD51 from cryo-EM structures.
Authors: 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 /
Abstract: 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.
History
DepositionJun 5, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 25, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 27, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

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Assembly

Deposited unit
D: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
A: Meiotic recombination protein DMC1/LIM15 homolog
B: Meiotic recombination protein DMC1/LIM15 homolog
C: Meiotic recombination protein DMC1/LIM15 homolog
hetero molecules


Theoretical massNumber of molelcules
Total (without water)117,52510
Polymers115,8864
Non-polymers1,6396
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: scanning transmission electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area13850 Å2
ΔGint-96 kcal/mol
Surface area39570 Å2

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Components

#1: DNA chain DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')


Mass: 2692.778 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: Protein Meiotic recombination protein DMC1/LIM15 homolog / Genetic recombination


Mass: 37731.031 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DMC1, DMC1H, LIM15 / Production host: Escherichia coli (E. coli) / Strain (production host): BLR / References: UniProt: Q14565
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Ca
#4: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM
Has ligand of interestN
Sequence detailsAuthors know the sequence of chains D: ...Authors know the sequence of chains D: TTATGTTCATTTTTTATATCCTTTACTTTATTTTCTCTGTTTATTCATTTACTTATTTTGTATTATCCTTATCTTATTTA. Since the DNA sequence could not be revealed by the helical reconstruction method, authors have used poly-T for model building.

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: DMC1-ssDNA filament / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Details: 25 mM Tris-HCl, pH 7.5, 50 mM KCl and 1 mM dithiothreitol) containing 2 mM AMP-PNP and 5 mM CaCl2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 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
Specimen supportGrid material: GRAPHENE OXIDE / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K
Details: 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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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 165000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Residual tilt: 10 mradians
Image recordingAverage exposure time: 5 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 30 eV
Image scansSampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

EM software
IDNameVersionCategory
2EPU2.2.0image acquisition
4SPHIRE3CTF correction
7PHENIX1.14model fitting
9Coot0.8.9.2model refinement
10PHENIX1.14model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 55.48 ° / Axial rise/subunit: 15.71 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 192466
3D reconstructionResolution: 3.33 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 71790 / Symmetry type: HELICAL

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