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

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.

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.

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 FSC_0.143 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 FSC_0.143 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 T_0-P_o, 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.

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, Rev7_B 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/Rev7_B interface whereas Site 2 highlights the ligand-binding site at Rev1 CTD/Rev7_B/Rev3_RIR 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 FSC_0.5 for each of the complexes are 3.8 Å and 3.1 Å, respectively.

Fig. 1 |. Architecture and subunit arrangement of the Rev1–Polζ holocomplex.

a, Schematic of the primary structure of S. cerevisiae Rev1 and Polζ holoenzyme subunits. Rev3, Pol31, Pol32 and Rev7 proteins are colored cyan, purple, salmon and green, respectively throughout the figure; Rev1 is yellow. PIP, PCNA-interacting protein; OB, oligonucleotide binding; PDE, phosphodiesterase. b, The cryo-EM density map of the Rev1–Polζ-DNA-dCTP holocomplex. DNA substrate is colored in red. The holocomplex is viewed perpendicular to the DNA axis from the front (left) as well as the rear (right) end of the ring. The N-helix-BRCT module is clearly visible from the rear view of the ring. c, Structural arrangement of the ternary Rev1–Polζ holocomplex as viewed from the same orientations as in b. The 4Fe–4S cluster is shown as a ruby cube.

Fig. 2 |. Rev1 N-helix and BRCT domain grasps the DNA.

a, The N-helix-BRCT module affixes to the major groove of the DNA. DNA is shown in light gray and the cartoon representation of Rev1 protein is shown in yellow. Key residues interacting with both the template and primer strands are highlighted. The location of G193 is shown; mutation of this residue (G193R; rev1–1) makes the cells defective in DNA damage response. b, Schematic representation of the N-helix-BRCT module interactions with DNA. Amino acid residues of the N-helix and BRCT domain are indicated and separated by brackets. DNA backbone phosphates are shown as red circles. Hydrogen bonds are indicated by purple lines (determined by interaction distance <3.6 Å); electrostatic interactions are shown by orange lines. The oligonucleotide numbers are highlighted adjacent to the identity of the bases. c, Structural organization of the Rev1 N-helix-BRCT module domain and the catalytic Rev3 subunit of Polζ encircling DNA. The location of the Rev1 BRCT domain is reminiscent of the PAD domain in Y-family polymerase and enmeshes within the palm (cyan), RIR (brown) and thumb domains (orange).

Fig. 3 |. Rev1 N-helix and BRCT domains slots in the palm, thumb and RIR domains of catalytic Rev3.

Structural organization of the Rev1 N-helix-BRCT module and Rev3, and detailed views of selected interaction sites and amino acid residues involved at the interfaces. Colors are as in Fig. 2c. The palm, thumb and RIR domains of Rev3 accommodate the Rev1 N-helix-BRCT module. a, Interactions between Rev1 BRCT and the NTD–palm linker as well as helices αG and α_XC of the palm domain via loops L1 and L3 of the BRCT domain. b, Elaboration of the interactions between the Rev1 N-helix and the NTD–palm linker and palm-loop within the palm domain of Rev3. The orientation has been rotated by 90° for a clear view of these interactions. c, Hydrophobic interface of Rev1 BRCT with the RIR and thumb domains of Rev3.

Fig. 4 |. Modeling of PCNA on the Rev1–Polζ holocomplex.

Structural alignment of the Rev1–Polζ holocomplex with the Polδ-PCNA complex (PDB 7KC0) indicates that PCNA can putatively interact with the C-terminal region of the Rev1 BRCT domain. IDCL, interdomain connector loop.

Fig. 5 |. Rev1-CTD forms a structured interface with Rev7_B, Pol32_N and the RIR domains of Rev3.

Overall structure of the region is shown in the center with Rev7_B colored in green, Rev1 colored in yellow and Rev3_RIR in brown. a, Detailed view of the interface between Rev1-CTD and the Pol32_N subunit. Selected amino acid residues of Pol32_N helix α1 and of Rev1-CTD helices α1 and α3 of the four helix bundle are shown. b, View of the hydrophobic interface formed between amino acid residues of the Rev1 C-terminal and N-terminal extended loops and the Rev7_B accessory subunit and RIR domain of Rev3. c, Detailed interactions of Rev7_B and the Rev1-CTD, primarily through the seatbelt region of Rev7_B.