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.

Rad53 limits CMG helicase uncoupling from DNA synthesis at replication forks

Subjects

Abstract

The coordination of DNA unwinding and synthesis at replication forks promotes efficient and faithful replication of chromosomal DNA. Disruption of the balance between helicase and polymerase activities during replication stress leads to fork progression defects and activation of the Rad53 checkpoint kinase, which is essential for the functional maintenance of stalled replication forks. The mechanism of Rad53-dependent fork stabilization is not known. Using reconstituted budding yeast replisomes, we show that mutational inactivation of the leading strand DNA polymerase, Pol ε, dNTP depletion, and chemical inhibition of DNA polymerases cause excessive DNA unwinding by the replicative DNA helicase, CMG, demonstrating that budding yeast replisomes lack intrinsic mechanisms that control helicase–polymerase coupling at the fork. Importantly, we find that the Rad53 kinase restricts excessive DNA unwinding at replication forks by limiting CMG helicase activity, suggesting a mechanism for fork stabilization by the replication checkpoint.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Active site mutations in the Pol ε polymerase domain inhibit DNA synthesis and induce excessive DNA unwinding at replication forks.
Fig. 2: Pol ε polymerase point mutants impede leading-strand synthesis in a concentration-dependent manner.
Fig. 3: PCNA binding and exonuclease activity of Pol2 are required for inhibition of Pol δ by Pol ε mutants.
Fig. 4: Inhibition of Pol ε competition with Pol δ suppresses the lethality of Pol2 polymerase active site mutations.
Fig. 5: Helicase–polymerase uncoupling at limiting dNTP concentrations.
Fig. 6: Rad53 inhibits DNA unwinding at replication forks after inhibition of DNA synthesis by aphidicolin.
Fig. 7: Rad53 control of DNA unwinding by CMG does not depend on Cdc45 or Mrc1.
Fig. 8: Rad53 control of DNA unwinding by CMG does not require inhibition of DNA synthesis.

Data availability

Source data for graphs in Figs. 2 and 3 and Extended Data Figs. 3 and 4 are available with the paper online.

References

  1. 1.

    Kim, S., Dallmann, H. G., McHenry, C. S. & Marians, K. J. Coupling of a replicative polymerase and helicase: a tau-DnaB interaction mediates rapid replication fork movement. Cell 84, 643–650 (1996).

    CAS  PubMed  Google Scholar 

  2. 2.

    Manosas, M., Spiering, M. M., Ding, F., Croquette, V. & Benkovic, S. J. Collaborative coupling between polymerase and helicase for leading-strand synthesis. Nucleic Acids Res. 40, 6187–6198 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Stano, N. M. et al. DNA synthesis provides the driving force to accelerate DNA unwinding by a helicase. Nature 435, 370–373 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Yeeles, J. T. P., Janska, A., Early, A. & Diffley, J. F. X. How the eukaryotic replisome achieves rapid and efficient DNA replication. Mol. Cell 65, 105–116 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Georgescu, R. E. et al. Mechanism of asymmetric polymerase assembly at the eukaryotic replication fork. Nat. Struct. Mol. Biol. 21, 664–670 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Taylor, M. R. G. & Yeeles, J. T. P. The initial response of a eukaryotic replisome to DNA damage. Mol. Cell 70, 1067–1080.e12 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Taylor, M. R. G. & Yeeles, J. T. P. Dynamics of replication fork progression following helicase–polymerase uncoupling in eukaryotes. J. Mol. Biol. 431, 2040–2049 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Walter, J. & Newport, J. Initiation of eukaryotic DNA replication: origin unwinding and sequential chromatin association of Cdc45, RPA, and DNA polymerase α. Mol Cell 5, 617–627 (2000).

    CAS  PubMed  Google Scholar 

  9. 9.

    Sparks, J. L. et al. The CMG helicase bypasses DNA-protein cross-links to facilitate their repair. Cell 176, 167–181.e21 (2019).

    CAS  PubMed  Google Scholar 

  10. 10.

    Gan, H. et al. Checkpoint Kinase Rad53 Couples Leading- and Lagging-Strand DNA Synthesis under Replication Stress. Mol. Cell 68, 446–455.e3 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Katou, Y. et al. S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex. Nature 424, 1078–1083 (2003).

    CAS  PubMed  Google Scholar 

  12. 12.

    Nedelcheva, M. N. et al. Uncoupling of unwinding from DNA synthesis implies regulation of MCM helicase by Tof1/Mrc1/Csm3 checkpoint complex. J. Mol. Biol. 347, 509–521 (2005).

    CAS  PubMed  Google Scholar 

  13. 13.

    Sabatinos, S. A., Green, M. D. & Forsburg, S. L. Continued DNA synthesis in replication checkpoint mutants leads to fork collapse. Mol. Cell. Biol. 32, 4986–4997 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Burnham, D. R., Kose, H. B., Hoyle, R. B. & Yardimci, H. The mechanism of DNA unwinding by the eukaryotic replicative helicase. Nat. Commun. 10, 2159 (2019).

    PubMed  PubMed Central  Google Scholar 

  15. 15.

    Graham, J. E., Marians, K. J. & Kowalczykowski, S. C. Independent and stochastic action of DNA polymerases in the replisome. Cell 169, 1201–1213.e17 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

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

  17. 17.

    Byun, T. S., Pacek, M., Yee, M. C., Walter, J. C. & Cimprich, K. A. Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint. Genes Dev. 19, 1040–1052 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Tercero, J. A., Longhese, M. P. & Diffley, J. F. A central role for DNA replication forks in checkpoint activation and response. Mol. Cell 11, 1323–1336 (2003).

    CAS  PubMed  Google Scholar 

  19. 19.

    Lopes, M. et al. The DNA replication checkpoint response stabilizes stalled replication forks. Nature 412, 557–561 (2001).

    CAS  PubMed  Google Scholar 

  20. 20.

    Sogo, J. M., Lopes, M. & Foiani, M. Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects. Science 297, 599–602 (2002).

    CAS  PubMed  Google Scholar 

  21. 21.

    De Piccoli, G. et al. Replisome stability at defective DNA replication forks is independent of S phase checkpoint kinases. Mol. Cell 45, 696–704 (2012).

    PubMed  PubMed Central  Google Scholar 

  22. 22.

    Dungrawala, H. et al. The replication checkpoint prevents two types of fork collapse without regulating replisome stability. Mol. Cell 59, 998–1010 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Szyjka, S. J. et al. Rad53 regulates replication fork restart after DNA damage in Saccharomyces cerevisiae. Genes Dev. 22, 1906–1920 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Iyer, D. R. & Rhind, N. Replication fork slowing and stalling are distinct, checkpoint-independent consequences of replicating damaged DNA. PLoS Genet. 13, e1006958 (2017).

    PubMed  PubMed Central  Google Scholar 

  25. 25.

    Poli, J. et al. dNTP pools determine fork progression and origin usage under replication stress. EMBO J. 31, 883–894 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Tercero, J. A. & Diffley, J. F. Regulation of DNA replication fork progression through damaged DNA by the Mec1/Rad53 checkpoint. Nature 412, 553–557 (2001).

    CAS  PubMed  Google Scholar 

  27. 27.

    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  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Bell, S. P. & Labib, K. Chromosome duplication in Saccharomyces cerevisiae. Genetics 203, 1027–1067 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Burgers, P. M. J. & Kunkel, T. A. Eukaryotic DNA replication fork. Annu. Rev. Biochem. 86, 417–438 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Tahirov, T. H., Makarova, K. S., Rogozin, I. B., Pavlov, Y. I. & Koonin, E. V. Evolution of DNA polymerases: an inactivated polymerase-exonuclease module in Pol ε and a chimeric origin of eukaryotic polymerases from two classes of archaeal ancestors. Biol. Direct 4, 11 (2009).

    PubMed  PubMed Central  Google Scholar 

  31. 31.

    Dua, R., Levy, D. L. & Campbell, J. L. Analysis of the essential functions of the C-terminal protein/protein interaction domain of Saccharomyces cerevisiae pol ε and its unexpected ability to support growth in the absence of the DNA polymerase domain. J. Biol. Chem. 274, 22283–22288 (1999).

    CAS  PubMed  Google Scholar 

  32. 32.

    Kesti, T., Flick, K., Keranen, S., Syvaoja, J. E. & Wittenberg, C. DNA polymerase ε catalytic domains are dispensable for DNA replication, DNA repair, and cell viability. Mol. Cell 3, 679–685 (1999).

    CAS  PubMed  Google Scholar 

  33. 33.

    Yu, C. et al. A mechanism for preventing asymmetric histone segregation onto replicating DNA strands. Science 361, 1386–1389 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Schauer, G. D. & O’Donnell, M. E. Quality control mechanisms exclude incorrect polymerases from the eukaryotic replication fork. Proc. Natl Acad. Sci. USA 114, 675–680 (2017).

    CAS  PubMed  Google Scholar 

  35. 35.

    Baker, T. A., Sekimizu, K., Funnell, B. E. & Kornberg, A. Extensive unwinding of the plasmid template during staged enzymatic initiation of DNA replication from the origin of the Escherichia coli chromosome. Cell 45, 53–64 (1986).

    CAS  PubMed  Google Scholar 

  36. 36.

    Dean, F. B. et al. Simian virus 40 (SV40) DNA replication: SV40 large T antigen unwinds DNA containing the SV40 origin of replication. Proc. Natl Acad. Sci. USA 84, 16–20 (1987).

    CAS  PubMed  Google Scholar 

  37. 37.

    Wold, M. S., Li, J. J. & Kelly, T. J. Initiation of simian virus 40 DNA replication in vitro: large-tumor-antigen- and origin-dependent unwinding of the template. Proc. Natl Acad. Sci. USA 84, 3643–3647 (1987).

    CAS  PubMed  Google Scholar 

  38. 38.

    Goswami, P. et al. Structure of DNA-CMG-Pol epsilon elucidates the roles of the non-catalytic polymerase modules in the eukaryotic replisome. Nat. Commun. 9, 5061 (2018).

    PubMed  PubMed Central  Google Scholar 

  39. 39.

    Langston, L. D. et al. CMG helicase and DNA polymerase epsilon form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication. Proc. Natl Acad. Sci. USA 111, 15390–15395 (2014).

    CAS  PubMed  Google Scholar 

  40. 40.

    Sun, J. et al. The architecture of a eukaryotic replisome. Nat. Struct. Mol. Biol. 22, 976–982 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Douglas, M. E., Ali, F. A., Costa, A. & Diffley, J. F. X. The mechanism of eukaryotic CMG helicase activation. Nature 555, 265–268 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Georgescu, R. E. et al. Reconstitution of a eukaryotic replisome reveals suppression mechanisms that define leading/lagging strand operation. Elife 4, e04988 (2015).

    PubMed  PubMed Central  Google Scholar 

  43. 43.

    Chilkova, O. et al. The eukaryotic leading and lagging strand DNA polymerases are loaded onto primer-ends via separate mechanisms but have comparable processivity in the presence of PCNA. Nucleic Acids Res. 35, 6588–6597 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Dua, R., Levy, D. L., Li, C. M., Snow, P. M. & Campbell, J. L. In vivo reconstitution of Saccharomyces cerevisiae DNA polymerase ε in insect cells. Purification and characterization. J. Biol. Chem. 277, 7889–7896 (2002).

    CAS  PubMed  Google Scholar 

  45. 45.

    Ganai, R. A., Bylund, G. O. & Johansson, E. Switching between polymerase and exonuclease sites in DNA polymerase epsilon. Nucleic Acids Res. 43, 932–942 (2015).

    CAS  PubMed  Google Scholar 

  46. 46.

    Garbacz, M. A. et al. Evidence that DNA polymerase δ contributes to initiating leading strand DNA replication in Saccharomyces cerevisiae. Nat. Commun. 9, 858 (2018).

    PubMed  PubMed Central  Google Scholar 

  47. 47.

    Acar, M., Becskei, A. & van Oudenaarden, A. Enhancement of cellular memory by reducing stochastic transitions. Nature 435, 228–232 (2005).

    CAS  PubMed  Google Scholar 

  48. 48.

    Szyjka, S. J., Viggiani, C. J. & Aparicio, O. M. Mrc1 is required for normal progression of replication forks throughout chromatin in S. cerevisiae. Mol. Cell 19, 691–697 (2005).

    CAS  PubMed  Google Scholar 

  49. 49.

    Tourriere, H., Versini, G., Cordon-Preciado, V., Alabert, C. & Pasero, P. Mrc1 and Tof1 promote replication fork progression and recovery independently of Rad53. Mol. Cell 19, 699–706 (2005).

    CAS  PubMed  Google Scholar 

  50. 50.

    Smolka, M. B., Albuquerque, C. P., Chen, S. H. & Zhou, H. Proteome-wide identification of in vivo targets of DNA damage checkpoint kinases. Proc. Natl Acad. Sci. USA 104, 10364–10369 (2007).

    CAS  PubMed  Google Scholar 

  51. 51.

    Alcasabas, A. A. et al. Mrc1 transduces signals of DNA replication stress to activate Rad53. Nat. Cell Biol. 3, 958–965 (2001).

    CAS  PubMed  Google Scholar 

  52. 52.

    Osborn, A. J. & Elledge, S. J. Mrc1 is a replication fork component whose phosphorylation in response to DNA replication stress activates Rad53. Genes Dev. 17, 1755–1767 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Can, G., Kauerhof, A. C., Macak, D. & Zegerman, P. Helicase subunit Cdc45 targets the checkpoint kinase Rad53 to both replication initiation and elongation complexes after fork stalling. Mol. Cell 73, 562–573.e3 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Langston, L. D. et al. Mcm10 promotes rapid isomerization of CMG-DNA for replisome bypass of lagging strand DNA blocks. Elife 6, e29118 (2017).

    PubMed  PubMed Central  Google Scholar 

  55. 55.

    Looke, M., Maloney, M. F. & Bell, S. P. Mcm10 regulates DNA replication elongation by stimulating the CMG replicative helicase. Genes Dev. 31, 291–305 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Burgers, P. M. Saccharomyces cerevisiae replication factor C. II. Formation and activity of complexes with the proliferating cell nuclear antigen and with DNA polymerases δ and ε. J. Biol. Chem. 266, 22698–22706 (1991).

    CAS  PubMed  Google Scholar 

  57. 57.

    Toledo, L. I. et al. ATR prohibits replication catastrophe by preventing global exhaustion of RPA. Cell 155, 1088–1103 (2013).

    CAS  PubMed  Google Scholar 

  58. 58.

    Gomez-Gonzalez, B., Patel, H., Early, A. & Diffley, J. F. X. Rpd3L contributes to the DNA damage sensitivity of Saccharomyces cerevisiae checkpoint mutants. Genetics 211, 503–513 (2019).

    CAS  PubMed  Google Scholar 

  59. 59.

    Ilves, I., Tamberg, N. & Botchan, M. R. Checkpoint kinase 2 (Chk2) inhibits the activity of the Cdc45/MCM2-7/GINS (CMG) replicative helicase complex. Proc. Natl Acad. Sci. USA 109, 13163–13170 (2012).

    CAS  PubMed  Google Scholar 

  60. 60.

    Gilbert, C. S., Green, C. M. & Lowndes, N. F. Budding yeast Rad9 is an ATP-dependent Rad53 activating machine. Mol. Cell 8, 129–136 (2001).

    CAS  PubMed  Google Scholar 

  61. 61.

    Gros, J., Devbhandari, S. & Remus, D. Origin plasticity during budding yeast DNA replication in vitro. EMBO J. 33, 621–636 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by NIH grant no. R01GM107239. We thank X. Zhao for yeast strains.

Author information

Affiliations

Authors

Contributions

D.R. and S.D. conceived this study and designed the experiments. S.D. performed the experiments. D.R. wrote the paper with help from S.D.

Corresponding author

Correspondence to Dirk Remus.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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.

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

Extended data

Extended Data Fig. 1 Reaction conditions affecting lagging strand synthesis in vitro.

a, Standard replication reactions were performed at various final salt concentrations as indicated. Template: pARS305. b, Titration of Pol δ into standard replication reactions. Template: pARS1. Reaction products were analyzed by 0.8 % alkaline agarose gel-electrophoresis and autoradiography. Uncropped gel images are available as source data.

Source data

Extended Data Fig. 2 Plasmid replication intermediates.

Leading strands are in red, lagging strands in blue. ERI: early replication intermediate; LRI: Late replication intermediate.

Extended Data Fig. 3 Characterization of replication products.

a, Standard replication reactions with pARS1 were carried out in the absence or presence of Top1 or Top2, as indicated. Reactions were stopped 60 minutes after origin activation, and replication products analyzed by native agarose gel-electrophoresis and autoradiography after linearization with the unique cutter Nde I, which cuts near the origin (fully replicated DNA molecules will resolve into linear monomers after linearization, whereas replication products containing unreplicated regions will resolve into double Y-shaped intermediates. Representative gel is shown on the left. The bar diagram depicts the average dissolution efficiency (fraction of linear full-length molecules per reaction) and s.d. of three independent experiments. b, Replication products from experiment in Fig. 1b were analyzed by alkaline agarose gel-electrophoresis and autoradiography. c, Pulse-chase experiment demonstrating that the ERI is a precursor of the LRI. Replisomes were formed on chromatin templates in the presence of α-32P-dCTP and stalled by omission of topoisomerase from the reaction (lane 1). After 15 minutes, Top1 was added to the reaction to release the stalled replisomes and intermediates chased by simultaneous addition of excess cold dCTP. At the indicated time points (lanes 2 and 3) replication products were isolated and analyzed by native (left) or denaturing agarose gel-electrophoresis and autoradiography. d, Standard replication reactions carried out in the presence of either Top1, Top2, or both. Replication products were analyzed by native agarose gel-electrophoresis and autoradiography. Uncropped gel images and data for graph in panel a are available as source data.

Source data

Extended Data Fig. 4 Reduced DNA synthesis and excess DNA unwinding in the presence of Pol ε polymerase mutants.

a, Total relative DNA synthesis in reactions of Fig. 1e were measured using ImageJ and plotted over time. b, Standard DNA replication reactions were carried out in the presence of wild-type or the indicated Pol ε variants (60 nM). 45 minutes after origin activation reactions were stopped and replication products analyzed by alkaline (left) or native (right) agarose gel-electrophoresis and autoradiography. Template: pARS1. c, Model for formation of θ and U* replication intermediates during plasmid replication in vitro. In normal DNA replication, the origin is initially unwound upon CMG activation (top left), followed shortly thereafter by the commencement of DNA synthesis and the coupling of leading strand synthesis to DNA unwinding by CMG (top center). Compensatory positive supercoils formed in the template during unwinding and fork progression are removed by Top1 and/or Top2. After deproteinization, the resulting θ structure is maintained (top right). In contrast, under conditions that slow-down DNA synthesis after origin unwinding the CMG helicase progresses along the template (bottom left) in advance of DNA synthesis; compensatory positive supercoils generated during DNA unwinding are removed by Top1 and/or Top2, and the unwound single-stranded DNA is stabilized by RPA binding (bottom center). Upon deproteinization, unwound complementary DNA strands reanneal, causing compensatory negative supercoils and thus resulting in a partially replicated, negatively supercoiled replication intermediate, U* (bottom right). Data for graph in panel a and uncropped gel images for b are available as source data.

Source data

Extended Data Fig. 5 Effect of Pol ε concentration on fork progression.

Pol εwt was titrated into standard replication reactions, reactions stopped 45 minutes after origin activation, and replication products analyzed by denaturing (left) or native (right) agarose gel-electrophoresis and autoradiography as indicated. Template: pARS1. Uncropped gel images are available as source data.

Source data

Extended Data Fig. 6 Primer extension by Pol δ and Pol ε.

a, Reaction scheme: Singly primed single-stranded M13mp18 DNA was pre-incubated with RPA, RFC/PCNA, three nucleotides, and Pol δ to initiate primer extension; Pol ε was subsequently added along with the remaining fourth nucleotide and incubation continued for 3 minutes. b, Denaturing agarose gel analysis of primer extension products obtained according to reaction scheme in a, but with either Pol δ or Pol ε omitted from the reaction. Uncropped gel images are available as source data.

Source data

Extended Data Fig. 7 Excessive DNA unwinding under limiting dNTP conditions in the absence of Csm3-Tof1-Mrc1 and presence of Pol εwt.

Pulse-chase experiment of standard replication reaction performed at 0.25 μM each dNTP as in Fig. 5b, with the following changes: 1) Pol εwt was used instead of Pol εexo-; 2) Csm3-Tof1 and Mrc1 were omitted from the reaction; 3) pARS305 instead of pARS1 served as a template. Time indicates minutes after origin activation. The reaction was chased with 500 μM cold dATP 5 minutes after origin activation. Reaction products were analyzed by agarose gel-electrophoresis and autoradiography. Uncropped gel images are available as source data.

Source data

Extended Data Fig. 8 Inhibition of DNA unwinding after CMG uncoupling from DNA synthesis is dependent on the kinase activity of Rad53.

a, Experiment demonstrating that U* DNA obtained in the presence of aphidicolin is negatively supercoiled. DNA isolated from the reaction analyzed in Fig. 6c, lane 5, was either mock-treated (lane 1) or treated with E. coli Topo I (lane 2) and analyzed by native agarose gel-electrophoresis. b, Purified wild-type and kinase-dead Rad53 (Rad53kd). Rad53-P: Autophosphorylated forms of Rad53. c, Effect of Rad53 kinase activity on U* formation after fork release from topological block in the presence of aphidicolin. Reactions were carried out as in Fig. 6c, except that wild-type (lanes 1-5) or kinase-dead (lanes 6-10) Rad53 was added to the reaction prior to fork release. Uncropped gel images are available as source data.

Source data

Extended Data Fig. 9 Inhibition of DNA unwinding after CMG uncoupling from DNA synthesis does not require CTM.

Experiment is as in Fig. 6c, except that Csm3-Tof1 and Mrc1 were omitted from the reaction. Uncropped gel images are available as source data.

Source data

Extended Data Fig. 10 Models for modes of fork progression in the presence of wild-type and catalytically dead Pol ε.

N and C indicate the N- or C-terminal exo-pol domain of Pol2. X indicates inactive mutant N-terminal Pol2 polymerase domain.

Supplementary information

Supplementary Information

Supplementary Note 1 and Tables 1–3.

Reporting Summary

Source data

Source Data Fig. 1

Uncropped gels.

Source Data Fig. 2

Uncropped gels.

Source Data Fig. 2

Source data for graphs.

Source Data Fig. 3

Uncropped gels.

Source Data Fig. 3

Source data for graphs.

Source Data Fig. 4

Uncropped gels.

Source Data Fig. 5

Uncropped gels.

Source Data Fig. 6

Uncropped gels.

Source Data Fig. 7

Uncropped gels.

Source Data Fig. 8

Uncropped gels.

Source Data Extended Data Fig. 1

Uncropped gels.

Source Data Extended Data Fig. 3

Uncropped gels.

Source Data Extended Data Fig. 3

Source data for graph in Extended Data Fig. 3a.

Source Data Extended Data Fig. 4

Uncropped gels.

Source Data Extended Data Fig. 4

Source data for graph in Extended Data Fig. 4a.

Source Data Extended Data Fig. 5

Uncropped gels.

Source Data Extended Data Fig. 6

Uncropped gels.

Source Data Extended Data Fig. 7

Uncropped gels.

Source Data Extended Data Fig. 8

Uncropped gels.

Source Data Extended Data Fig. 9

Uncropped gels.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Devbhandari, S., Remus, D. Rad53 limits CMG helicase uncoupling from DNA synthesis at replication forks. Nat Struct Mol Biol 27, 461–471 (2020). https://doi.org/10.1038/s41594-020-0407-7

Download citation

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