DNA polymerase ζ (Polζ) belongs to the same B-family as high-fidelity replicative polymerases, yet is specialized for the extension reaction in translesion DNA synthesis (TLS). Despite its importance in TLS, the structure of Polζ is unknown. We present cryo-EM structures of the Saccharomyces cerevisiae Polζ holoenzyme in the act of DNA synthesis (3.1 Å) and without DNA (4.1 Å). Polζ displays a pentameric ring-like architecture, with catalytic Rev3, accessory Pol31‚ Pol32 and two Rev7 subunits forming an uninterrupted daisy chain of protein–protein interactions. We also uncover the features that impose high fidelity during the nucleotide-incorporation step and those that accommodate mismatches and lesions during the extension reaction. Collectively, we decrypt the molecular underpinnings of Polζ’s role in TLS and provide a framework for new cancer therapeutics.
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We thank B. Carragher, C. Potter and E. Eng for helpful advice and discussion throughout the project. We also thank Z. Zhang and D. Bobe for help in grid preparation, Y. Z. Tan for help in collecting tilted cryo-EM data, A. Brown and T. Terwilliger for help in implementing software and D. Nair for help in model building. This work was primarily funded by grant R01-GM124047 from the National Institutes of Health (NIH). I.U.-B. was supported by a grant PID2019-104423GB-I00/AEI/10.13039/501100011033 from the Spanish State Research Agency and by the Basque Excellence Research Centre program. Initial EM screening was performed at the Icahn School of Medicine microscope facility supported by a shared instrumentation grant from the NIH (1S10RR026473). Most of the cryo-EM work was performed at the Simons Electron Microscopy Center and National Resource for Automated Molecular Microscopy, located at the New York Structural Biology Center, supported by grants from the Simons Foundation (SF349247), NYSTAR and the NIH National Institute of General Medical Sciences (GM103310), with additional support from Agouron Institute (F00316), NIH (OD019994) and NIH (RR029300). Computing resources needed for this work were provided in part by the High Performance Computing facility of the Icahn School of Medicine at Mount Sinai. Molecular graphics and analyses were performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311.
The authors declare no competing interests.
Peer review information Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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a, Data collected at 0° stage angle resulted in disproportionally low number of classes for side-views of the ternary complex of Polζ depicted in the 2D class averages. This resulted in a ‘smeared 3D model’ as shown by the anisotropic 3DFSC plot. Scale bar = 123 Å. b, Data collected at a stage angle of 0° for the apo-state of Polζ also had preferred set of views as shown in the 2D class averages. The final construction was anisotropic as depicted by the directional FSC plot. Scale bar = 123 Å.
a, Data were collected on Chameleon grids and particles from one session were picked with template based picker (FindEM) and processed in cryoSPARC to give a consensus map with a FSC0.143 of 3.57 Å. Major stages of processing are shown schematically and particles involved at each stage are highlighted in green. Scale bar = 137 Å. b, Final particles from two sessions were merged and used to train Topaz. Data processing from Topaz picked particles in cryoSPARC2 improved the sphericity. A schematic representation of the improved consensus map displaying a FSC0.143 of 3.2 Å is shown. Scale bar = 137 Å. c, Focused refinement of the final volume was done in cryoSPARC2. Masks were created (along the blue dashed line) for 3D refinement of Rev3 and accessory subunits separately to give consensus maps at 3.02 Å and 3.08 Å, respectively.
a, Data were collected at a 40° tilt angle and processed in cryoSPARC to give a good distribution of particles (green) with different views depicted in the 2D class averages. The final 3D reconstruction displaying a FSC0.143 of 4.1 Å showed an isotropic map amenable for model building. Scale bar = 123 Å. b, Per- particle CTF refinement of the map improved the sphericity further as shown by the 3DFSC plot.
The NTD in Rev3 and Pol3, is composed of three motifs (I, II, III) but is much more elaborate and extended in Rev3. Loop 1 and Loop 2 contact all three motifs and connect the NTD to the fingers and palm domains, respectively.
Residues around the T1 site are shown (sticks) for Rev3 (left) and Pol3 (right). Surface for the palm domains and DNA are shown in cyan and grey, respectively. The T1 base (red) and the key residues are highlighted in dots.
a, Sequence alignment of yeast and human RBM1 and RBM2 regions of Rev3. Conserved prolines within RBM1 and RBM2 are highlighted in green. Also, highlighted are the conserved residues among the yeast and human homologs within the RIR region. b, Structural comparison of the yeast and human RBM1 and RBM2. Individual structures of human Rev7 with RBM1 peptide (hRev7:RBM1; PDB ID: 3ABD) and RBM2 peptide (hRev7:RBM2; PDB ID: 6BC8) are compared to the corresponding sub-regions (yRev7A:RBM1; yRev7B:RBM2) in the yeast Polζ holoenzyme. The protein residues involved in the interactions are highlighted in green and the RIR is shown in brown. The interactions of Rev7A and Rev7B with the RIR segment connecting RBM1 and RBM2 (yRev7A:yRev7B:RIRint) is also depicted.
A superimposition of the CysBD of the Polζ (left; grey in color) and Polδ (right; yellow in color; PDB ID: 6P1H) shows conservation in its overall topology. Notably, helix αXM in Polζ CysBD has been substituted by a loop in Polδ (PDB ID: 6P1H). All the four cysteines interacting with the 4Fe−4S cluster in Rev3 are also highlighted.
Overlay of the palm domains of Rev3 and Pol II show a similar trajectory for the NTD-palm linker. In Rev3, this trajectory is coupled to interactions with the Palm-loop. The Pol II template DNA strand is shown in yellow (PDB ID: 3K5M). A close-up view of the looped-out abasic lesion and the adjoining 5′ guanine nucleotide. Notably, the guanine base clashes with the backbone carbonyl of E954 in Rev3.
a, Superimposition of human Rev7-RBM1-Rev1CTD (PDB ID: 4EXT) on Rev7B shows close proximity to Pol32N (shown in yellow), highlighting the importance of Pol32N in stabilizing this interaction. b, Superimposition of the human Rev7-RBM1-Rev1CTD on Rev7A shows clashes of Rev1CTD with various secondary structure elements of Rev7B (shown in yellow).
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Malik, R., Kopylov, M., Gomez-Llorente, Y. et al. Structure and mechanism of B-family DNA polymerase ζ specialized for translesion DNA synthesis. Nat Struct Mol Biol 27, 913–924 (2020). https://doi.org/10.1038/s41594-020-0476-7
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