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- EMDB-29746: Cryo-EM structure of the Mismatch Uncoupling Complex (II) of Huma... -
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Basic information
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Title | Cryo-EM structure of the Mismatch Uncoupling Complex (II) of Human Mitochondrial DNA Polymerase Gamma | |||||||||
![]() | Cryo-EM structure of the Mismatch Uncoupling Complex (II) of Human Mitochondrial DNA Polymerase Gamma | |||||||||
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![]() | Mitochondrial DNA Polymerase / DNA Proofreading / PolG / REPLICATION / REPLICATION-DNA complex | |||||||||
Function / homology | ![]() gamma DNA polymerase complex / mitochondrial DNA replication / positive regulation of DNA-directed DNA polymerase activity / single-stranded DNA 3'-5' DNA exonuclease activity / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA metabolic process / DNA replication proofreading / DNA polymerase processivity factor activity / mitochondrial nucleoid / Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases ...gamma DNA polymerase complex / mitochondrial DNA replication / positive regulation of DNA-directed DNA polymerase activity / single-stranded DNA 3'-5' DNA exonuclease activity / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA metabolic process / DNA replication proofreading / DNA polymerase processivity factor activity / mitochondrial nucleoid / Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases / 5'-deoxyribose-5-phosphate lyase activity / DNA polymerase binding / 3'-5' exonuclease activity / base-excision repair, gap-filling / Transcriptional activation of mitochondrial biogenesis / base-excision repair / DNA-templated DNA replication / protease binding / double-stranded DNA binding / in utero embryonic development / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / mitochondrial matrix / intracellular membrane-bounded organelle / chromatin binding / protein-containing complex / mitochondrion / DNA binding / identical protein binding / cytoplasm Similarity search - Function | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.63 Å | |||||||||
![]() | Nayak AR / Buchel G / Herbine KH / Sarfallah A / Sokolova VO / Zamudio-Ochoa A / Temiakov D | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Structural basis for DNA proofreading. Authors: Gina Buchel / Ashok R Nayak / Karl Herbine / Azadeh Sarfallah / Viktoriia O Sokolova / Angelica Zamudio-Ochoa / Dmitry Temiakov / ![]() Abstract: DNA polymerase (DNAP) can correct errors in DNA during replication by proofreading, a process critical for cell viability. However, the mechanism by which an erroneously incorporated base ...DNA polymerase (DNAP) can correct errors in DNA during replication by proofreading, a process critical for cell viability. However, the mechanism by which an erroneously incorporated base translocates from the polymerase to the exonuclease site and the corrected DNA terminus returns has remained elusive. Here, we present an ensemble of nine high-resolution structures representing human mitochondrial DNA polymerase Gamma, Polγ, captured during consecutive proofreading steps. The structures reveal key events, including mismatched base recognition, its dissociation from the polymerase site, forward translocation of DNAP, alterations in DNA trajectory, repositioning and refolding of elements for primer separation, DNAP backtracking, and displacement of the mismatched base into the exonuclease site. Altogether, our findings suggest a conserved 'bolt-action' mechanism of proofreading based on iterative cycles of DNAP translocation without dissociation from the DNA, facilitating primer transfer between catalytic sites. Functional assays and mutagenesis corroborate this mechanism, connecting pathogenic mutations to crucial structural elements in proofreading steps. | |||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 118 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 21 KB 21 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 10.6 KB | Display | ![]() |
Images | ![]() | 157.8 KB | ||
Filedesc metadata | ![]() | 7.7 KB | ||
Others | ![]() ![]() | 10.3 MB 10.3 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8g5jMC ![]() 8g5iC ![]() 8g5kC ![]() 8g5lC ![]() 8g5mC ![]() 8g5nC ![]() 8g5oC ![]() 8g5pC ![]() 8t7eC M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | Cryo-EM structure of the Mismatch Uncoupling Complex (II) of Human Mitochondrial DNA Polymerase Gamma | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.826 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: Half Map 1
File | emd_29746_half_map_1.map | ||||||||||||
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Annotation | Half Map 1 | ||||||||||||
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Density Histograms |
-Half map: Half Map 2
File | emd_29746_half_map_2.map | ||||||||||||
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Annotation | Half Map 2 | ||||||||||||
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Density Histograms |
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Sample components
-Entire : Cryo-EM structure of the Mismatch Uncoupling Complex (II) of Huma...
Entire | Name: Cryo-EM structure of the Mismatch Uncoupling Complex (II) of Human Mitochondrial DNA Polymerase Gamma |
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Components |
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-Supramolecule #1: Cryo-EM structure of the Mismatch Uncoupling Complex (II) of Huma...
Supramolecule | Name: Cryo-EM structure of the Mismatch Uncoupling Complex (II) of Human Mitochondrial DNA Polymerase Gamma type: complex / ID: 1 / Parent: 0 / Macromolecule list: all Details: Human mitochondrial DNA polymerase PolG (wild type) assembled on a DNA-DNA scaffold |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 362 KDa |
-Macromolecule #1: DNA polymerase subunit gamma-1
Macromolecule | Name: DNA polymerase subunit gamma-1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 139.730703 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MSRLLWRKVA GATVGPGPVP APGRWVSSSV PASDPSDGQR RRQQQQQQQQ QQQQQPQQPQ VLSSEGGQLR HNPLDIQMLS RGLHEQIFG QGGEMPGEAA VRRSVEHLQK HGLWGQPAVP LPDVELRLPP LYGDNLDQHF RLLAQKQSLP YLEAANLLLQ A QLPPKPPA ...String: MSRLLWRKVA GATVGPGPVP APGRWVSSSV PASDPSDGQR RRQQQQQQQQ QQQQQPQQPQ VLSSEGGQLR HNPLDIQMLS RGLHEQIFG QGGEMPGEAA VRRSVEHLQK HGLWGQPAVP LPDVELRLPP LYGDNLDQHF RLLAQKQSLP YLEAANLLLQ A QLPPKPPA WAWAEGWTRY GPEGEAVPVA IPEERALVFD VEVCLAEGTC PTLAVAISPS AWYSWCSQRL VEERYSWTSQ LS PADLIPL EVPTGASSPT QRDWQEQLVV GHNVSFDRAH IREQYLIQGS RMRFLDTMSM HMAISGLSSF QRSLWIAAKQ GKH KVQPPT KQGQKSQRKA RRGPAISSWD WLDISSVNSL AEVHRLYVGG PPLEKEPREL FVKGTMKDIR ENFQDLMQYC AQDV WATHE VFQQQLPLFL ERCPHPVTLA GMLEMGVSYL PVNQNWERYL AEAQGTYEEL QREMKKSLMD LANDACQLLS GERYK EDPW LWDLEWDLQE FKQKKAKKVK KEPATASKLP IEGAGAPGDP MDQEDLGPCS EEEEFQQDVM ARACLQKLKG TTELLP KRP QHLPGHPGWY RKLCPRLDDP AWTPGPSLLS LQMRVTPKLM ALTWDGFPLH YSERHGWGYL VPGRRDNLAK LPTGTTL ES AGVVCPYRAI ESLYRKHCLE QGKQQLMPQE AGLAEEFLLT DNSAIWQTVE ELDYLEVEAE AKMENLRAAV PGQPLALT A RGGPKDTQPS YHHGNGPYND VDIPGCWFFK LPHKDGNSCN VGSPFAKDFL PKMEDGTLQA GPGGASGPRA LEINKMISF WRNAHKRISS QMVVWLPRSA LPRAVIRHPD YDEEGLYGAI LPQVVTAGTI TRRAVEPTWL TASNARPDRV GSELKAMVQA PPGYTLVGA DVDSQELWIA AVLGDAHFAG MHGCTAFGWM TLQGRKSRGT DLHSKTATTV GISREHAKIF NYGRIYGAGQ P FAERLLMQ FNHRLTQQEA AEKAQQMYAA TKGLRWYRLS DEGEWLVREL NLPVDRTEGG WISLQDLRKV QRETARKSQW KK WEVVAER AWKGGTESEM FNKLESIATS DIPRTPVLGC CISRALEPSA VQEEFMTSRV NWVVQSSAVD YLHLMLVAMK WLF EEFAID GRFCISIHDE VRYLVREEDR YRAALALQIT NLLTRCMFAY KLGLNDLPQS VAFFSAVDID RCLRKEVTMD CKTP SNPTG MERRYGIPQG EALDIYQIIE LTKGSLEKRS QPGP UniProtKB: DNA polymerase subunit gamma-1 |
-Macromolecule #2: DNA polymerase subunit gamma-2, mitochondrial
Macromolecule | Name: DNA polymerase subunit gamma-2, mitochondrial / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 54.991 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MRSRVAVRAC HKVCRCLLSG FGGRVDAGQP ELLTERSSPK GGHVKSHAEL EGNGEHPEAP GSGEGSEALL EICQRRHFLS GSKQQLSRD SLLSGCHPGF GPLGVELRKN LAAEWWTSVV VFREQVFPVD ALHHKPGPLL PGDSAFRLVS AETLREILQD K ELSKEQLV ...String: MRSRVAVRAC HKVCRCLLSG FGGRVDAGQP ELLTERSSPK GGHVKSHAEL EGNGEHPEAP GSGEGSEALL EICQRRHFLS GSKQQLSRD SLLSGCHPGF GPLGVELRKN LAAEWWTSVV VFREQVFPVD ALHHKPGPLL PGDSAFRLVS AETLREILQD K ELSKEQLV AFLENVLKTS GKLRENLLHG ALEHYVNCLD LVNKRLPYGL AQIGVCFHPV FDTKQIRNGV KSIGEKTEAS LV WFTPPRT SNQWLDFWLR HRLQWWRKFA MSPSNFSSSD CQDEEGRKGN KLYYNFPWGK ELIETLWNLG DHELLHMYPG NVS KLHGRD GRKNVVPCVL SVNGDLDRGM LAYLYDSFQL TENSFTRKKN LHRKVLKLHP CLAPIKVALD VGRGPTLELR QVCQ GLFNE LLENGISVWP GYLETMQSSL EQLYSKYDEM SILFTVLVTE TTLENGLIHL RSRDTTMKEM MHISKLKDFL IKYIS SAKN V UniProtKB: DNA polymerase subunit gamma-2 |
-Macromolecule #3: Mismatched Primer DNA
Macromolecule | Name: Mismatched Primer DNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 6.209018 KDa |
Sequence | String: (DG)(DA)(DA)(DG)(DA)(DC)(DA)(DG)(DT)(DC) (DT)(DG)(DC)(DG)(DG)(DC)(DG)(DC)(DG)(DA) |
-Macromolecule #4: Template DNA
Macromolecule | Name: Template DNA / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 7.894083 KDa |
Sequence | String: (DA)(DC)(DA)(DC)(DA)(DC)(DG)(DC)(DG)(DC) (DG)(DC)(DC)(DG)(DC)(DA)(DG)(DA)(DC)(DT) (DG)(DT)(DC)(DT)(DT)(DC) |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.52 mg/mL |
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Buffer | pH: 7.9 Details: 10 mM Tris-Hcl pH 7.9, 100 mM Nacl, 10 mM DTT, and 2 mM MgCl2 |
Grid | Model: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
Details | 2 uM complex of the wild type PolG and DNA-DNA scaffold containing a mismatched base |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 2 / Number real images: 10572 / Average electron dose: 70.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 18.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000 |
Sample stage | Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
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Output model | ![]() PDB-8g5j: |