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- PDB-7cgy: Human DMC1 Q244M mutant of the post-synaptic complexes -

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Basic information

Entry
Database: PDB / ID: 7cgy
TitleHuman DMC1 Q244M mutant of the post-synaptic complexes
ComponentsMeiotic recombination protein DMC1/LIM15 homologGenetic recombination
KeywordsRECOMBINATION / meiotic homologous recombination / DNA repair / ATPase
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 / Meiotic recombination protein DMC1/LIM15 homolog
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsChi, H.Y. / Ho, M.C. / Tsai, M.D. / Luo, S.C. / Yeh, H.Y.
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
DepositionJul 3, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 18, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 25, 2020Group: Structure summary / Category: audit_author
Revision 1.2Mar 17, 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
Revision 1.3Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
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)114,8419
Polymers113,2023
Non-polymers1,6396
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: scanning transmission electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area10470 Å2
ΔGint-68 kcal/mol
Surface area40880 Å2

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Components

#1: Protein Meiotic recombination protein DMC1/LIM15 homolog / Genetic recombination


Mass: 37734.098 Da / Num. of mol.: 3 / Mutation: Q244M
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
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Ca
#3: 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

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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

Component
IDNameTypeEntity IDParent-IDSourceDetails
1DMC1Q244M-dsDNA filamentCOMPLEX#10RECOMBINANT
2DMC1 Q244M mutantCOMPLEX#11RECOMBINANT
3homologous double strand DNACOMPLEX#11NATURALdensity map of DNA is missing duo to the flexible/dynamic property of dsDNA in the DMC1 Q244M filament
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 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 / 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 scansWidth: 3838 / Height: 3710 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

EM software
IDNameVersionCategory
1RELION3particle selection
2EPU2.2.0image acquisition
4RELION3CTF correction
7PHENIX1.14model fitting
10Coot0.8.9.2model refinement
11Rosetta3.9model refinement
15RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 55.93 ° / Axial rise/subunit: 16 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 643100
Details: 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.
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 232018 / Algorithm: FOURIER SPACE / Num. of class averages: 4 / Symmetry type: HELICAL

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