Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Mechanisms of loading and release of the 9-1-1 checkpoint clamp

Abstract

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.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: Structure of Rad24-RFC in complex with 9-1-1 and a 5′ ss/dsDNA junction.
Fig. 2: Coordination of the 5′ ss/dsDNA junction by Rad24-RFC.
Fig. 3: Comparison of open and closed checkpoint clamp states.
Fig. 4: Rad24 binds to Ddc1 and Mec3.
Fig. 5: ATP hydrolysis by Rad24-RFC disrupts the 9-1-1 and DNA binding interfaces.
Fig. 6: Model for loading and release of the 9-1-1 checkpoint clamp by Rad24-RFC.

Data availability

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.

References

  1. Ciccia, A. & Elledge, S. J. The DNA damage response: making it safe to play with knives. Mol. Cell 40, 179–204 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. Pardo, B., Crabbe, L. & Pasero, P. Signaling pathways of replication stress in yeast. FEMS Yeast Res. 17, https://doi.org/10.1093/femsyr/fow101 (2017).

  3. Saldivar, J. C., Cortez, D. & Cimprich, K. A. The essential kinase ATR: ensuring faithful duplication of a challenging genome. Nat. Rev. Mol. Cell Biol. 18, 622–636 (2017).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. Ellison, V. & Stillman, B. Biochemical characterization of DNA damage checkpoint complexes: clamp loader and clamp complexes with specificity for 5′ recessed DNA. PLoS Biol. 1, E33 (2003).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Majka, J., Binz, S. K., Wold, M. S. & Burgers, P. M. Replication protein A directs loading of the DNA damage checkpoint clamp to 5′-DNA junctions. J. Biol. Chem. 281, 27855–27861 (2006).

    CAS  Article  PubMed  Google Scholar 

  6. Zou, L. Four pillars of the S-phase checkpoint. Genes Dev. 27, 227–233 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Gobbini, E., Casari, E., Colombo, C. V., Bonetti, D. & Longhese, M. P. The 9-1-1 complex controls Mre11 nuclease and checkpoint activation during short-range resection of DNA double-strand breaks. Cell Rep. 33, 108287 (2020).

    CAS  Article  PubMed  Google Scholar 

  8. Ngo, G. H. P., Balakrishnan, L., Dubarry, M., Campbell, J. L. & Lydall, D. The 9-1-1 checkpoint clamp stimulates DNA resection by Dna2-Sgs1 and Exo1. Nucleic Acids Res. 42, 10516–10528 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. van Schendel, R., Romeijn, R., Buijs, H. & Tijsterman, M. Preservation of lagging strand integrity at sites of stalled replication by Pol α-primase and 9-1-1 complex. Sci. Adv. 7, eabf2278 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Dore, A. S., Kilkenny, M. L., Rzechorzek, N. J. & Pearl, L. H. Crystal structure of the rad9-rad1-hus1 DNA damage checkpoint complex–implications for clamp loading and regulation. Mol. Cell 34, 735–745 (2009).

    CAS  Article  PubMed  Google Scholar 

  11. Sohn, S. Y. & Cho, Y. Crystal structure of the human rad9-hus1-rad1 clamp. J. Mol. Biol. 390, 490–502 (2009).

    CAS  Article  PubMed  Google Scholar 

  12. Xu, M. et al. Structure and functional implications of the human Rad9-Hus1-Rad1 cell cycle checkpoint complex. J. Biol. Chem. 284, 20457–20461 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. Bermudez, V. P. et al. Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro. Proc. Natl Acad. Sci. USA 100, 1633–1638 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. Majka, J. & Burgers, P. M. Yeast Rad17/Mec3/Ddc1: a sliding clamp for the DNA damage checkpoint. Proc. Natl Acad. Sci. USA 100, 2249–2254 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Lee, K. Y. & Park, S. H. Eukaryotic clamp loaders and unloaders in the maintenance of genome stability. Exp. Mol. Med. 52, 1948–1958 (2020).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. Kelch, B. A. Review: The lord of the rings: structure and mechanism of the sliding clamp loader. Biopolymers 105, 532–546 (2016).

    CAS  Article  PubMed  Google Scholar 

  17. Bowman, G. D., O’Donnell, M. & Kuriyan, J. Structural analysis of a eukaryotic sliding DNA clamp-clamp loader complex. Nature 429, 724–730 (2004).

    CAS  Article  PubMed  Google Scholar 

  18. Gaubitz, C. et al. Structure of the human clamp loader reveals an autoinhibited conformation of a substrate-bound AAA+ switch. Proc. Natl Acad. Sci. USA 117, 23571–23580 (2020).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Devbhandari, S., Jiang, J., Kumar, C., Whitehouse, I. & Remus, D. Chromatin constrains the initiation and elongation of DNA replication. Mol. Cell 65, 131–141 (2017).

    CAS  Article  PubMed  Google Scholar 

  20. Kelch, B. A., Makino, D. L., O’Donnell, M. & Kuriyan, J. How a DNA polymerase clamp loader opens a sliding clamp. Science 334, 1675–1680 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. Michael, W. M., Ott, R., Fanning, E. & Newport, J. Activation of the DNA replication checkpoint through RNA synthesis by primase. Science 289, 2133–2137 (2000).

    CAS  Article  PubMed  Google Scholar 

  22. MacDougall, C. A., Byun, T. S., Van, C., Yee, M. C. & Cimprich, K. A. The structural determinants of checkpoint activation. Genes Dev. 21, 898–903 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. Van, C., Yan, S., Michael, W. M., Waga, S. & Cimprich, K. A. Continued primer synthesis at stalled replication forks contributes to checkpoint activation. J. Cell Biol. 189, 233–246 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. Baretic, D. et al. Cryo-EM structure of the fork protection complex bound to CMG at a replication fork. Mol. Cell 78, 926–940 e913 (2020).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. Gao, Y. et al. Structures and operating principles of the replisome. Science https://doi.org/10.1126/science.aav7003 (2019).

  26. Lee, J. Y. & Yang, W. UvrD helicase unwinds DNA one base pair at a time by a two-part power stroke. Cell 127, 1349–1360 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. Majka, J., Chung, B. Y. & Burgers, P. M. Requirement for ATP by the DNA damage checkpoint clamp loader. J. Biol. Chem. 279, 20921–20926 (2004).

    CAS  Article  PubMed  Google Scholar 

  28. Naiki, T., Shimomura, T., Kondo, T., Matsumoto, K. & Sugimoto, K. Rfc5, in cooperation with rad24, controls DNA damage checkpoints throughout the cell cycle in Saccharomyces cerevisiae. Mol. Cell. Biol. 20, 5888–5896 (2000).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. Majka, J., Niedziela-Majka, A. & Burgers, P. M. The checkpoint clamp activates Mec1 kinase during initiation of the DNA damage checkpoint. Mol. Cell 24, 891–901 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. Kazmirski, S. L., Zhao, Y., Bowman, G. D., O’Donnell, M. & Kuriyan, J. Out-of-plane motions in open sliding clamps: molecular dynamics simulations of eukaryotic and archaeal proliferating cell nuclear antigen. Proc. Natl Acad. Sci. USA 102, 13801–13806 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. Miyata, T. et al. Open clamp structure in the clamp-loading complex visualized by electron microscopic image analysis. Proc. Natl Acad. Sci. USA 102, 13795–13800 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article  PubMed  Google Scholar 

  33. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    CAS  Article  PubMed  Google Scholar 

  34. Scheres, S. H. Processing of structurally heterogeneous cryo-EM sata in RELION. Methods Enzymol. 579, 125–157 (2016).

    CAS  Article  PubMed  Google Scholar 

  35. Punjani, A., Zhang, H. & Fleet, D. J. Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction. Nat. Methods 17, 1214–1221 (2020).

    CAS  Article  PubMed  Google Scholar 

  36. Terwilliger, T. C., Ludtke, S. J., Read, R. J., Adams, P. D. & Afonine, P. V. Improvement of cryo-EM maps by density modification. Nat. Methods 17, 923–927 (2020).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    CAS  Article  PubMed  Google Scholar 

  38. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D. Biol. Crystallogr. 66, 486–501 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. Croll, T. I. ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps. Acta Crystallogr. D. Struct. Biol. 74, 519–530 (2018).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D. Struct. Biol. 75, 861–877 (2019).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Contributions

J.C.C., M.S., D.R. and R.K.H. conceptualized, performed and analyzed the experiments. J.C.C. purified all proteins. M.S. collected and processed cryo-EM data. M.S and R.K.H. built models. All authors contributed to writing the paper.

Corresponding authors

Correspondence to Dirk Remus or Richard K. Hite.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Structural and Molecular Biology thanks David Jeruzalmi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. 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. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Purification and analysis of Rad24-RFC:9-1-1.

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.

Source data

Extended Data Fig. 2 Validation of Rad24-RFC:9-1-1 structures.

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.

Extended Data Fig. 3 Comparison of Rad24-RFC:9-1-1 with RFC-PCNA.

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.

Extended Data Fig. 4 Coordination of DNA by Rad24-RFC:9-1-1.

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.

Extended Data Fig. 5 Comparison of open and closed states of Rad24-RFC:9-1-1.

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).

Extended Data Fig. 6 Validation of Rad24-RFCADP structure.

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.

Extended Data Fig. 7 Conformational changes induced by ATP hydrolysis in Rad24-RFC.

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 information

Supplementary Information

Supplementary Tables 1–3.

Reporting Summary

Peer Review Information

Supplementary Video 1

Morph between open and closed states of Rad24-RFC–9-1-1.

Supplementary Video 2

Morph of 9-1-1 in the closed and open states of Rad24-RFC–9-1-1.

Supplementary Video 3

Morph of Rad24-RFC between ATP-bound state in Rad24-RFC–9-1-1 and ADP-bound state in Rad24-RFCADP.

Source data

Source Data Extended Data Fig. 1

Source data for ATPase assay.

Source Data Extended Data Fig. 1

Unprocessed SDS–PAGE.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41594-022-00741-7

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing