Accurate replication of mitochondrial DNA (mtDNA) by DNA polymerase γ (Polγ) is essential for maintaining cellular energy supplies, metabolism, and cell cycle control. To illustrate the structural mechanism for Polγ coordinating polymerase (pol) and exonuclease (exo) activities to ensure rapid and accurate DNA synthesis, we determined four cryo-EM structures of Polγ captured after accurate or erroneous incorporation to a resolution of 2.4–3.0 Å. The structures show that Polγ employs a dual-checkpoint mechanism to sense nucleotide misincorporation and initiate proofreading. The transition from replication to error editing is accompanied by increased dynamics in both DNA and enzyme, in which the polymerase relaxes its processivity and the primer–template DNA unwinds, rotates, and backtracks to shuttle the mismatch-containing primer terminus 32 Å to the exo site for editing. Our structural and functional studies also provide a foundation for analyses of Polγ mutation-induced human diseases and aging.
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Cryo-EM density maps and corresponding coordinates have been deposited in the Worldwide Protein Data Bank (PDB) OneDep System under the following accession codes: G-C replication complex (EMD-27154) (PDB 8D33), G•T R conformer (EMD-27155) (PDB 8D37), G•T I conformer (EMD-27163) (PDB 8D3R), and G•T E conformer (EMD-27172) (PDB 8D42). Accession codes for consensus and local refinement maps of the G•T E conformer are EMD-27169 (consensus), EMD-27170 (local refinement of subunit A and p/t DNA) and EMD-27171 (local refinement of subunit B).
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We thank W. Chiu for scientific and technical insights and helpful discussions, K.-Y. Wong for expert computational support and M. Mayer for preliminary data collection at the Stanford-SLAC Cryo-EM Center (S2C2) supported by the National Institutes of Health Common Fund (U24 GM129541). We thank M. Gagnon, P. Leiman and K. Kaus for critical reading of the manuscript. The work is supported by an NIH grant (R01 AI134611) to Y.W.Y., the James W. McLaughlin Fellowship Fund to J.P. and an endowment from the Sealy and Smith Foundation to the Sealy Center for Structural Biology and Molecular Biophysics at UTMB.
The authors declare no competing interests.
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Extended Data Fig. 1 Cryo-EM single-particle data processing pipeline for replication ternary complex.
a, Data were imported into cryoSPARC for 2D and 3D image analysis. See Methods for details. b, EM map of replication complex colored by local resolution estimated by cryoSPARC’s implementation of blocres at 0.5 FSC threshold and FSC resolution at 0.143 GSFSC threshold reported by cryoSPARC.
Extended Data Fig. 2 Cryo-EM single-particle data processing pipeline for proofreading ternary complex.
a, Data were imported into cryoSPARC for 2D and 3D image analysis. See Methods for details. b, EM map of proofreading E-conformer (Left), I-conformer (Middle), and R-conformer (Right) colored by local resolution estimated by cryoSPARC’s implementation of blocres at 0.5 FSC threshold and FSC resolution at 0.143 GSFSC threshold reported by cryoSPARC.
Extended Data Fig. 3 Superposition of G·C replication complex and G·T mismatch R-conformer in the pol active site.
Superposition of (a) DNA, catalytic residue (Asp1135), and RQH triad (Arg853Gln1102His1134), (b) trigger loop, and (c) catalytic loop and Fingers subdomain. G·C replication complex and G·T R-conformer are shown in transparent and light green, respectively. Primer strand is shown in pink, template strand in gray, and mismatched nucleoside in red. Overlay of primer-template base pair in the P-site with corresponding EM density in (d) G·C replication complex and in (e) G·T R-conformer. (f) Superposition of 3'-end of the primer in the pol active site of G·C replication complex (transparent) and G·T R-conformer (light green) showing the positioning of 3'-OH. Residues Asp890, Asp1135, Arg853, and His1134 as well as the incoming nucleotide, dCTP, are displayed.
Incoming nucleotide dCTP is in the pol active site of (a) the replication complex and (b-d) proofreading complex. In R-conformers (a, b), dCTP forms W-C pair with the template but does not form any base pair in the I-conformer (c) and in the E-conformer (d). Electron density for dCTP and Ca2+ shown in light blue mesh. Proteins are colored based on their conformation, primer strand in pink, template strand in gray, and mismatched nucleoside in red. e, Scheme of DNA synthesis pathway. Fidelity checkpoint at pre- and post-nucleotide incorporation stages in Pol γ. In the N-site, where incoming nucleotide binds, Watson-Crick base checking first chooses the correct nucleotide triphosphate to incorporate. After the incorporation, Arg853 and Gln1102 checks the nascent base pair for correct Watson-Crick geometry.
Extended Data Fig. 5 Catalytic loop positioning and trigger loop interaction in Pol γ ternary complexes.
a–c, Comparison of catalytic loops between G·C replicating complex and G·T R-conformer (a), I-conformer (b), and G·T E-conformer (c). d–f, Detailed interaction between trigger loop and primer strand in R- (d), I- (e), and E-conformers (f). g–i, Superposition of trigger loops in three proofreading structures. Primer strand from R- (g), I- (h), and E- (i) conformers are shown.
a-c, Superposition of Fingers subdomain (a), Thumb subdomain (b), and polymerase and exonuclease active sites (c) of proofreading structures and apo Pol γ (tan). Proofreading structures are colored according to conformation: R (light green), I (white), and E (light blue). d, Superimposed subunit interface of E-conformer and the apo enzyme.
a-c, Overall DNA movement associated with conformational changes are shown between R- and I- (a), I- and E- (b), and R- and E-conformers (c). d, Superposition of the primer strand in the correctly matched G·C replication complex and mismatch G·T E-conformer after superimposing the invariable exo active site, nucleoside n-5 is marked as a point of reference for comparison. e, Triangular primer 3′-end shuttling from the pol site to the exo site via an intermediate of the pathway. The primer moves 9.5 Å from R-conformer pol site to I-conformer and another 33.9 Å to the exo site in the E-conformer to shuttle between pol and exo sites that are 32.7 Å apart. Catalytic residues for pol (green) and exo (light blue) are shown.
Extended Data Fig. 8 DNA position in proofreading structures relative to exonuclease site and splitter helix.
Top (left) and 90° rotated (right) views of R- (a), I- (b), and E- (c) conformers. In R-conformer (a), DNA sits in polymerase active site and does not contact splitter helix. In I-conformer (b), DNA is lifted out of the polymerase active site, but does not reach the splitter helix. In E-conformer (c), DNA is completely out of the polymerase active site and is stabilized by the splitter helix at the fork junction.
a-d, Side-by-side comparison of polymerase active site of Pol γ G·C replication complex (a), Taq Klenow Fragment (PDB: 3KTQ) (b), RB69 DNA polymerase (PDB: 3NCI) (c), and Taq DNA polymerase III α subunit (PDB: 3E0D) (d) from A-,B-, and C-family DNA polymerases, respectively. e-h, Side-by-side comparison of DNA fork junction in Pol γ G·T E-conformer (e), E. coli Klenow Fragment (PDB: 1KLN) (f), RB69 DNA polymerase (PDB: 1CLQ) (g), and E. coli DNA polymerase III (PDB: 5M1S) (h) from A-,B-, and C-family DNA polymerases, respectively.
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Park, J., Herrmann, G.K., Mitchell, P.G. et al. Polγ coordinates DNA synthesis and proofreading to ensure mitochondrial genome integrity. Nat Struct Mol Biol 30, 812–823 (2023). https://doi.org/10.1038/s41594-023-00980-2