Single-stranded or double-stranded DNA junctions with recessed 5′ ends serve as loading sites for the checkpoint clamp, 9-1-1, which mediates activation of the apical checkpoint kinase, ATRMec1. However, the basis for 9-1-1’s recruitment to 5′ junctions is unclear. Here, we present structures of the yeast checkpoint clamp loader, Rad24-replication factor C (RFC), in complex with 9-1-1 and a 5′ junction and in a post-ATP-hydrolysis state. Unexpectedly, 9-1-1 adopts both closed and planar open states in the presence of Rad24-RFC and DNA. Moreover, Rad24-RFC associates with the DNA junction in the opposite orientation of processivity clamp loaders with Rad24 exclusively coordinating the double-stranded region. ATP hydrolysis stimulates conformational changes in Rad24-RFC, leading to disengagement of DNA-loaded 9-1-1. Together, these structures explain 9-1-1’s recruitment to 5′ junctions and reveal new principles of sliding clamp loading.
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Cryo-EM maps and atomic coordinates have been deposited with the Electron Microscopy Data Bank and PDB under accession codes EMD-25422 and PDB 7ST9 for Rad24-RFC–9-1-1 in the open state, code EMD-25424 for the Mec3 focus refinement of Rad24-RFC–9-1-1 in the open state, codes EMD-25423 and PDB 7STB for Rad24-RFC–9-1-1 in the closed state, codes EMDB-25426 and PDB 7STE for Rad24-RFCADP, and code EMD-25425 for the Rad24 AAA+ focus refinement of Rad24-RFCADP. Datasets from PDB used in this study include 1SXJ and 3A1J. Source data are provided with this paper.
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We thank M. de la Cruz at the Memorial Sloan Kettering Cancer Center (MSKCC) Richard Rifkind Center for cryo-EM for assistance with data collection and the MSKCC HPC group for assistance with data processing. This work was supported by NIH-NCI Cancer Center Support grant nos. P30 CA008748 (D.R. and R.K.H.), NIGMS R01-GM107239 (D.R.) and NIGMS R01-GM127428 (D.R.), and the Josie Robertson Investigators Program (R.K.H.). M.S. is a Walter Benjamin Fellow of the Deutsche Forschungsgemeinschaft.
The authors declare no competing interests.
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a, Representative Coomassie-stained SDS-PAGE analysis of purified S. cerevisiae Rad24-RFC, 9-1-1 and RPA. The * corresponds to truncated form of Rad24. b, ATPase activity of Rad24-RFC in the presence of 9-1-1 and DNA (black circles), Rad24-RFC in the presence of DNA (teal triangles), Rad24-RFC in the presence of 9-1-1 (purple triangles) and 9-1-1 and DNA (pink squares). Experiments are shown in triplicate. c, Schematic for the assembly of Rad24-RFC:9-1-1:DNA in the presence of ATPγS. d, Representative silver-stained SDS-PAGE analysis of Rad24-RFC:9-1-1 fractions following glycerol gradient (10-35%) centrifugation. Fractions pooled for Cryo-EM analysis are denoted with *. e, Representative Coomassie-stained SDS-PAGE analysis of purified S. cerevisiae Rad24-RFC:9-1-1:DNA. Fractionation experiments shown in panels d and e have been repeated at least three times.
a, Representative cryo-EM image of vitrified Rad24-RFC:9-1-1:DNA. b, Representative two-dimensional averages of Rad24-RFC:9-1-1. c, Plot of Fourier shell correlations between two independent open state half-maps (blue), two independent closed state half-maps (red), two independent Mec3 focus half-maps (brown), the open state map and open state atomic model (cyan), and the closed state map and closed state atomic model (magenta). d-f Cryo-EM density maps of open (d), Mec3-focused refined (e) and closed (f) maps colored by local resolution in Å. Region included in the mask for the Mec3 focused refinement (e) is denoted by the dashed line in d.
a-b, Structures of Rad24-RFC:9-1-1 (a) and RFC-PCNA (b, PDB:1SXJ) colored by subunit with Rad24-RFC / RFC in surface depiction and 9-1-1 / PCNA in ribbon depiction. The channel formed between the AAA + , collar and A’ domains of Rad24 in a is highlighted by the dashed oval. c, Superposition of Rad24-RFC (colored by domain) with RFC (gray). The models are aligned by the collar domains. d, Superposition of Rad24-RFC subunits (colored by subunit) with RFC subunits (grey). The subunits are aligned by their collar domains. For Rad24 and Rfc1, the A’ domains are removed for clarity. e, Nucleotide-binding sites in the AAA + domains Rad24-RFC:9-1-1. Nucleotides and residues whose side chains coordinate the nucleotide are shown in sticks.
a, Rad24-RFC:9-1-1 surface colored by electrostatic surface potential. DNA is shown as cartoon and Rad24 channel is highlighted by the dashed oval. b, Interactions between ssDNA backbone and conserved arginine residues in Rfc2-5 establish the spiral B-form like conformation of the ssDNA. Arginine side chains are shown as spheres and Rad24 is removed for clarity. c, Superposition of Rad24 in open Rad24-RFC:9-1-1 (colored by subunit) with Rfc1 in RFC-PCNA (colored in grey, PDB:1SXJ), aligned by the AAA + domains. Single-stranded DNA is shown as cartoon. Dashed oval highlights insertion in the AAA + domain of Rad24 that occludes double-stranded DNA from occupying the central cavity of Rad24-RFC. Rfc2-5 are removed for clarity. d, Alignment of residues that coordinate the 5’ junction in S. cerevisiae Rad24 with Rad17 from X. laevis, M. musculus, and H. sapiens. Arrows denote conserved Phe340, which serves as the pin, and conserved Lys345, which coordinates the 5’ phosphate. e, Alignment of residues that coordinate the single-stranded DNA in S. cerevisiae Rfc2, Rfc3 and Rfc4 with RFC4, RFC5 and RFC2 from H. sapiens. Arrows denote conserved isoleucine and arginine residues that coordinate the DNA backbone.
a, Superposition of open (colored by subunit) and closed states of Rad24-RFC:9-1-1 (colored in grey), shown as two views. b, Structure of 9-1-1 in the open state of Rad24-RFC:9-1-1, colored by RMSD in Å. RMSD is calculated on per-subunit basis between the open and closed states of Rad24-RFC:9-1-1. c-d Superposition of 9-1-1 clamp in Rad24-RFC:9-1-1 in closed (c) and open (d) states (colored by subunit) with a structure of human 9-1-1 clamp (colored in grey, PDB:3A1J).
a, Representative cryo-EM image of vitrified Rad24-RFC in the presence of ADP. b, Representative two-dimensional averages of Rad24-RFCADP. c, Plot of Fourier shell correlations between two independent consensus half-maps (blue), two independent Rad24 focus refinement half-maps (brown), and the consensus map and model (cyan). d-e, Cryo-EM density maps of consensus (d) and Rad24 focus refinement (e) maps colored by local resolution in Å. Region included in the mask for the Rad24 AAA + focused refinement (e) is denoted by the dashed line in d.
a, Superposition of Rad24-RFC in Rad24-RFCADP (colored by subunit) and Rad24-RFC:9-1-1 (colored in grey) shown in two views. b, Nucleotide-binding sites in the AAA + domains of Rad24-RFCADP. Nucleotides are shown as sticks. c, Superposition of the AAA + inter-domain domain interfaces in Rad24-RFCADP (colored by subunit) and Rad24-RFC:9-1-1 (colored in grey). Nucleotides are shown as sticks.
Supplementary Tables 1–3.
Morph between open and closed states of Rad24-RFC–9-1-1.
Morph of 9-1-1 in the closed and open states of Rad24-RFC–9-1-1.
Morph of Rad24-RFC between ATP-bound state in Rad24-RFC–9-1-1 and ADP-bound state in Rad24-RFCADP.
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Castaneda, J.C., Schrecker, M., Remus, D. et al. Mechanisms of loading and release of the 9-1-1 checkpoint clamp. Nat Struct Mol Biol 29, 369–375 (2022). https://doi.org/10.1038/s41594-022-00741-7
Nature Structural & Molecular Biology (2022)