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Cryo-EM structure of the Rev1–Polζ holocomplex reveals the mechanism of their cooperativity in translesion DNA synthesis

Nature Structural & Molecular Biology (2024)Cite this article

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Abstract

Rev1–Polζ-dependent translesion synthesis (TLS) of DNA is crucial for maintaining genome integrity. To elucidate the mechanism by which the two polymerases cooperate in TLS, we determined the cryogenic electron microscopic structure of the Saccharomyces cerevisiae Rev1–Polζ holocomplex in the act of DNA synthesis (3.53 Å). We discovered that a composite N-helix-BRCT module in Rev1 is the keystone of Rev1–Polζ cooperativity, interacting directly with the DNA template–primer and with the Rev3 catalytic subunit of Polζ. The module is positioned akin to the polymerase-associated domain in Y-family TLS polymerases and is set ideally to interact with PCNA. We delineate the full extent of interactions that the carboxy-terminal domain of Rev1 makes with Polζ and identify potential new druggable sites to suppress chemoresistance from first-line chemotherapeutics. Collectively, our results provide fundamental new insights into the mechanism of cooperativity between Rev1 and Polζ in TLS.

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Fig. 1: Architecture and subunit arrangement of the Rev1–Polζ holocomplex.
Fig. 2: Rev1 N-helix and BRCT domain grasps the DNA.
Fig. 3: Rev1 N-helix and BRCT domains slots in the palm, thumb and RIR domains of catalytic Rev3.
Fig. 4: Modeling of PCNA on the Rev1–Polζ holocomplex.
Fig. 5: Rev1-CTD forms a structured interface with Rev7B, Pol32N and the RIR domains of Rev3.

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Data availability

The data that support this study are available from the corresponding author upon reasonable request. The cryo-EM density maps of Rev1-Full–Polζ-DNA-dCTP and Rev1-ΔN–Polζ-DNA-dCTP generated in this study have been deposited in the Electron Microscopy Data Bank (EMDB) under accession numbers EMD-41371 and EMD-41372, respectively. The resulting atomic coordinates for Rev1-Full–Polζ-DNA-dCTP and Rev1-ΔN–Polζ-DNA-dCTP have been deposited in the Protein Data Bank (PDB) with accession numbers PDB 8TLQ and PDB 8TLT, respectively. Atomic coordinates from previously published work used in the study are available in the PDB with accession numbers PDB 6V93 and PDB 4ID3. Source data are provided with this paper.

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Acknowledgements

This work was funded by grant R35-GM131780 (to A.K.A) from the National Institutes of Health (NIH). I.U.-B. acknowledges support from the Spanish Ministry of Science, Innovation and Universities. Cryo-EM data collection 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). We thank J. Wang, J. Mendez and E. T. Eng for assistance with cryo-EM data collection. 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.

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Authors and Affiliations

  1. Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Radhika Malik & Aneel K. Aggarwal

  2. Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA

    Robert E. Johnson, Louise Prakash & Satya Prakash

  3. Instituto Biofisika (UPV/EHU, CSIC), University of the Basque Country, Leioa, Spain

    Iban Ubarretxena-Belandia

  4. Ikerbasque Basque Foundation for Science, Bilbao, Spain

    Iban Ubarretxena-Belandia

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  1. Radhika Malik
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Contributions

R.M., A.K.A., R.E.J. and S.P. designed the experiments; R.E.J. characterized, expressed and assembled the Rev1–Polζ complexes in yeast; R.M. purified the Rev1–Polζ complexes and performed all the cryo-EM studies. R.M. built, characterized and refined the atomic models. A.K.A. guided the overall project; S.P. and L.P. guided the biochemical and protein expression studies. A.K.A., R.M., R.E.J., S.P. and L.P. prepared the manuscript with input from I.U.-B.

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Correspondence to Radhika Malik or Aneel K. Aggarwal.

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Extended data

Extended Data Fig. 1 Purification of a Rev1-Polζ holoenzyme complex.

a, Superdex 200 PC3.2/30 Gel filtration purification of the Rev1-Polζ holocomplex. A summary of the purification steps up to the gel filtration step are shown. 1 μl each from fractions 7-20 were separated on a 4-20% gradient SDS-PAGE gel. Molecular weight marker sizes are shown on the left and protein identities are shown on the right. b, Purified Rev1-Polζ. Fractions 8-10 from the gel filtration step (panel A) containing stoichiometric amounts of each subunit of Rev1-Polζ were pooled and incubated with 200 μl Glutathione Sepharose beads to remove any residual prescission protease and the resulting protein preparation was concentrated using a microcon MWCO-100 concentrator. Lane 1, 1.0 μl Rev1-Polζ holocomplex. Molecular weight marker sizes are shown on the left and protein identities are shown on the right. c, SDS gel profile of Rev1-ΔN-Polζ. Coomassie stained 4-20% SDS PAGE of purified Rev1-ΔN-Polζ holocomplex employed for vitrification. d, SDS gel profile of Rev1-Full-Polζ. Coomassie blue stained 4-20% SDS PAGE of purified Rev1-Full-Polζ holocomplex employed for vitrification. All the gels were run twice. Subunit identities and molecular weight markers are shown for each gel.

Source data

Extended Data Fig. 2 Cryo-EM data processing of the Rev1-ΔN-Polζ holocomplex.

a, Representative electron micrograph is shown. Scale bar is 200 nm. 8353 micrographs were collected and processed for the cryo-EM reconstruction b, An overview of the workflow for the cryo-EM data processing is shown. Schematic shows particle picking using blob picker and Topaz. Various stages of data processing includes 2-D classification and Ab-initio clean-up using cryoSPARC. The representative 2-D classes are shown and the major 3-D class is used for refinement. The map calculated after non-uniform refinement has a FSC0.143 of 2.85 Å. Number of Particles used at key stages of data processing are shown in blue. c, Cryo-EM density map of the Rev1-ΔN-Polζ holocomplex colored by local resolution is shown. Scale bar is in Å.

Extended Data Fig. 3 Cryo-EM data processing of the Rev1-Full-Polζ holocomplex.

a, Representative electron micrograph is shown. Scale bar is 200 nm. 13,404 micrographs were collected and processed for the cryo-EM reconstruction. b, An overview of the workflow for the cryo-EM data processing shows particle picking using Topaz and processing including 2-D classification and Ab-initio reconstruction using cryoSPARC. The representative 2-D classes are shown and the major 3-D class is used for refinement. The map calculated after non-uniform refinement has a FSC0.143 of 3.53 Å. Number of particles at key stages of data processing are highlighted in blue. c, Cryo-EM density map of the Rev1-Full-Polζ holocomplex colored by local resolution. Scale bar is in Å.

Extended Data Fig. 4 Catalytic Rev3 structure in the Rev1-Polζ holocomplex.

a, Structure of Rev3 colored by domain is shown. Dark blue, brown, magenta, cyan, yellow, orange and gray denote, respectively, the N-terminal, RIR, exo, palm, fingers, thumb and C-terminal domains of Rev3. The DNA is highlighted in red. b, Cryo-EM density of the T0-Po, T1-P1 and T2-P2 bases of the DNA for Rev1-Full-Polζ (left) as well as Rev1-ΔN-Polζ (right) holocomplexes.

Extended Data Fig. 5 The assembly of Rev1 into the Polζ holocomplex in yeast cells depends upon Rev1 BRCT.

Polζ complexes were purified by α-Flag affinity purification of Flag-tagged Rev3 from protein extracts of yeast cells overexpressing individual subunits of the Rev1-Full-Polζ holocomplex and analyzed by SDS-PAGE and Coomassie staining. The complexes were eluted with prescission protease, and free GST and prescission protease were subsequently removed by incubation with Glutathione Sepharose. Lane 1, Rev1-Polζ holocomplex purified from cells expressing wild type Rev3/Rev7/Pol31/Pol32/Rev1 subunits. Rev1 copurifies with the Polζ holoenzyme. Lane 2, Polζ complex from cells as in lane 1, but lacking overexpression of the Pol32 subunit. Rev1 copurifies with the Polζ complex, despite the lack of Pol32. Lane 3, Rev1-Polζ holocomplex from cells as in lane 1, but overexpressing the rev1-1 mutant protein which harbors the G193R mutation within the BRCT domain. The rev1-1 mutant does not copurify with the Polζ complex. Expression of the rev1-1 protein was confirmed by Western blotting of total protein extract using affinity purified α-Rev1 antibodies. The gel was run once. The Protein identities and molecular weight sizes (kDa) are shown on the right and left, respectively.

Source data

Extended Data Fig. 6 Overlay of the palm domains of the Rev1-Polζ holocomplex and Pol3 highlighting the outward conformation of the NTD-palm linker in Rev3.

Superimposition of the palm domains of Rev3 and Pol3 shows the NTD-palm linker of Pol3 trekking a path closer to the template DNA (shown in red; T0 and T1 bases are highlighted) in comparison to Rev3.

Extended Data Fig. 7 Comparison of surface representation of BRCT domains of Rev1 with PARP1, RFC, and BRCA1.

a, Structural alignment of the BRCT domains of Rev1, PARP1, RFC and BRCA1. b, Electrostatic surface potential of the Rev1 N-helix-BRCT module shows an extensive positively charged surface (blue) for binding DNA (red). c, d, Electrostatic surface potentials of the PARP1 (c) and RFC BRCT (d) domains are also positively charged (blue) for interacting with DNA (red) e, Electrostatic surface potential of the BRCA1 BRCT1 differs from that of Rev1/PARP1/RFC BRCT domains.

Extended Data Fig. 8 Sequence alignment of BRCT domains from various proteins.

Amino acid sequence alignment of conventional as well as DNA binding BRCT domains is shown. Equivalent residues among all the BRCTs are highlighted in yellow. Secondary structure elements of the Rev1 N-helix-BRCT module are shown above and are derived from the coordinates of Rev1-Polζ holocomplex structure (PDB ID: 8TLQ) using ESpript. Helices are designated as black coils and beta sheets are indicated by blue arrows. Key DNA binding residues in Rev1 BRCT are highlighted by a purple star and those conserved between the yeast and human Rev1 BRCT are highlighted in red.

Extended Data Fig. 9 Druggable ligand-binding sites predicted for the Rev1 CTD/Polζ interface.

Rev1 CTD, Rev7B and a portion of the Rev3-RIR are shown in yellow, green and brown, respectively. Potential ligand binding Site 1 and Site 2, as predicted by FTsite, are shown in salmon and grey colored mesh, respectively. The cluster of probes at each site are shown in sticks. Site 1 represents the Rev1 CTD/Rev7B interface whereas Site 2 highlights the ligand-binding site at Rev1 CTD/Rev7B/Rev3RIR interface.

Extended Data Fig. 10 Model-map FSC curves.

The curves for Rev1-Full-Polζ (a) as well as Rev1-ΔN-Polζ (b) holocomplexes are shown where the FSC0.5 for each of the complexes are 3.8 Å and 3.1 Å, respectively.

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Source Data file for Extended Data Fig. 1

Source Data Extended Data Fig. 5

Source Data file for Extended Data Fig. 5

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Malik, R., Johnson, R.E., Ubarretxena-Belandia, I. et al. Cryo-EM structure of the Rev1–Polζ holocomplex reveals the mechanism of their cooperativity in translesion DNA synthesis. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01302-w

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