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Reconstitution of translesion synthesis reveals a mechanism of eukaryotic DNA replication restart

Nature Structural & Molecular Biology volume 27, pages 450–460 (2020)Cite this article

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Abstract

Leading-strand template aberrations cause helicase–polymerase uncoupling and impede replication fork progression, but the details of how uncoupled forks are restarted remain uncertain. Using purified proteins from Saccharomyces cerevisiae, we have reconstituted translesion synthesis (TLS)-mediated restart of a eukaryotic replisome following collision with a cyclobutane pyrimidine dimer. We find that TLS functions ‘on the fly’ to promote resumption of rapid replication fork rates, despite lesion bypass occurring uncoupled from the Cdc45-MCM-GINS (CMG) helicase. Surprisingly, the main lagging-strand polymerase, Pol δ, binds the leading strand upon uncoupling and inhibits TLS. Pol δ is also crucial for efficient recoupling of leading-strand synthesis to CMG following lesion bypass. Proliferating cell nuclear antigen monoubiquitination positively regulates TLS to overcome Pol δ inhibition. We reveal that these mechanisms of negative and positive regulation also operate on the lagging strand. Our observations have implications for both fork restart and the division of labor during leading-strand synthesis generally.

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Fig. 1: Reconstitution of lagging- and leading-strand TLS.
Fig. 2: On-the-fly TLS occurs uncoupled from CMG.
Fig. 3: Pol δ, but not Pol ε, inhibits lagging and leading-strand TLS.
Fig. 4: Pol δ recouples leading-strand synthesis to CMG following TLS.
Fig. 5: PCNA monoubiquitination stimulates on-the-fly TLS.
Fig. 6: PCNA monoubiquitination promotes lagging-strand TLS.
Fig. 7: Model of leading- and lagging-strand TLS by Pol η.

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Data availability

All data are provided in full in the Results section and the Supplementary Information accompanying this paper. Unprocessed gels are available with the paper online.

References

  1. Daigaku, Y. et al. A global profile of replicative polymerase usage. Nat. Struct. Mol. Biol. 22, 192–198 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. McElhinny, N. S. A., Gordenin, D. A., Stith, C. M., Burgers, P. M. J. & Kunkel, T. A. Division of labor at the eukaryotic replication fork. Mol. Cell 30, 137–144 (2008).

    Article  CAS  Google Scholar 

  3. Pursell, Z. F., Isoz, I., Lundström, E.-B., Johansson, E. & Kunkel, T. A. Yeast DNA polymerase ε participates in leading-strand DNA replication. Science 317, 127–130 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Zeman, M. K. & Cimprich, K. A. Causes and consequences of replication stress. Nat. Cell Biol. 16, 2–9 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Marians, K. J. Lesion bypass and the reactivation of stalled replication forks. Annu. Rev. Biochem. 87, 217–238 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Sale, J. E., Lehmann, A. R. & Woodgate, R. Y-family DNA polymerases and their role in tolerance of cellular DNA damage. Nat. Rev. Mol. Cell Biol. 13, 141–152 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. McCulloch, S. D. & Kunkel, T. A. The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesis polymerases. Cell Res. 18, 148–161 (2008).

    Article  CAS  PubMed  Google Scholar 

  8. Leung, W., Baxley, R. M., Moldovan, G.-L. & Bielinsky, A.-K. Mechanisms of DNA damage tolerance: post-translational regulation of PCNA. Genes 10, 10 (2019).

    Article  CAS  Google Scholar 

  9. Bienko, M. et al. Ubiquitin-binding domains in Y-family polymerases regulate translesion synthesis. Science 310, 1821–1824 (2005).

    Article  CAS  PubMed  Google Scholar 

  10. Plosky, B. S. et al. Controlling the subcellular localization of DNA polymerases ι and η via interactions with ubiquitin. EMBO J. 25, 2847–2855 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Hoege, C., Pfander, B., Moldovan, G.-L., Pyrowolakis, G. & Jentsch, S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419, 135–141 (2002).

    Article  CAS  Google Scholar 

  12. Stelter, P. & Ulrich, H. D. Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation. Nature 425, 188–191 (2003).

    Article  CAS  Google Scholar 

  13. Garg, P. & Burgers, P. M. Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases η and REV1. Proc. Natl Acad. Sci. USA 102, 18361–18366 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Haracska, L., Unk, I., Prakash, L. & Prakash, S. Ubiquitylation of yeast proliferating cell nuclear antigen and its implications for translesion DNA synthesis. Proc. Natl Acad. Sci. USA 103, 6477–6482 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Acharya, N. et al. Roles of PCNA-binding and ubiquitin-binding domains in human DNA polymerase η in translesion DNA synthesis. Proc. Natl Acad. Sci. USA 105, 17724–17729 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Acharya, N., Brahma, A., Haracska, L., Prakash, L. & Prakash, S. Mutations in the ubiquitin binding UBZ motif of DNA polymerase η do not impair its function in translesion synthesis during replication. Mol. Cell. Biol. 27, 7266–7272 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Parker, J. L., Bielen, A. B., Dikic, I. & Ulrich, H. D. Contributions of ubiquitin- and PCNA-binding domains to the activity of polymerase η in Saccharomyces cerevisiae. Nucleic Acids Res. 35, 881–889 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Acharya, N. et al. Reply to Sabbioneda et al.: role of ubiquitin-binding motif of human DNA polymerase η in translesion synthesis. Proc. Natl Acad. Sci. USA 106, E21 (2009).

    CAS  PubMed Central  Google Scholar 

  19. Arakawa, H. et al. A role for PCNA ubiquitination in immunoglobulin hypermutation. PLoS Biol. 4, e366 (2006).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Despras, E., Delrieu, N., Garandeau, C., Ahmed‐Seghir, S. & Kannouche, P. L. Regulation of the specialized DNA polymerase η: revisiting the biological relevance of its PCNA- and ubiquitin-binding motifs. Environ. Mol. Mutagen. 53, 752–765 (2012).

    Article  CAS  PubMed  Google Scholar 

  21. Göhler, T., Sabbioneda, S., Green, C. M. & Lehmann, A. R. ATR-mediated phosphorylation of DNA polymerase η is needed for efficient recovery from UV damage. J. Cell Biol. 192, 219–227 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Hendel, A. et al. PCNA ubiquitination is important, but not essential for translesion DNA synthesis in mammalian cells. PLoS Genet. 7, e1002262 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Krijger, P. H. L. et al. PCNA ubiquitination-independent activation of polymerase η during somatic hypermutation and DNA damage tolerance. DNA Repair 10, 1051–1059 (2011).

    Article  CAS  PubMed  Google Scholar 

  24. Sabbioneda, S. et al. Effect of proliferating cell nuclear antigen ubiquitination and chromatin structure on the dynamic properties of the Y-family DNA polymerases. Mol. Biol. Cell 19, 5193–5202 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Edmunds, C. E., Simpson, L. J. & Sale, J. E. PCNA ubiquitination and REV1 define temporally distinct mechanisms for controlling translesion synthesis in the avian cell line DT40. Mol. Cell 30, 519–529 (2008).

    Article  CAS  PubMed  Google Scholar 

  26. Lopes, M., Foiani, M. & Sogo, J. M. Multiple mechanisms control chromosome integrity after replication fork uncoupling and restart at irreparable UV lesions. Mol. Cell 21, 15–27 (2006).

    Article  CAS  PubMed  Google Scholar 

  27. Daigaku, Y., Davies, A. A. & Ulrich, H. D. Ubiquitin-dependent DNA damage bypass is separable from genome replication. Nature 465, 951–955 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Karras, G. I. & Jentsch, S. The RAD6 DNA damage tolerance pathway operates uncoupled from the replication fork and is functional beyond S phase. Cell 141, 255–267 (2010).

    Article  CAS  PubMed  Google Scholar 

  29. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Yeeles, J. T. P., Deegan, T. D., Janska, A., Early, A. & Diffley, J. F. X. Regulated eukaryotic DNA replication origin firing with purified proteins. Nature 519, 431–435 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  35. Aria, V. & Yeeles, J. T. P. Mechanism of bidirectional leading-strand synthesis establishment at eukaryotic DNA replication origins. Mol. Cell 73, 199–211 (2019).

    Article  CAS  PubMed Central  Google Scholar 

  36. 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Zhou, Z.-X., Lujan, S. A., Burkholder, A. B., Garbacz, M. A. & Kunkel, T. A. Roles for DNA polymerase δ in initiating and terminating leading strand DNA replication. Nat. Commun. 10, 3992 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Becker, J. R. et al. Genetic interactions implicating postreplicative repair in Okazaki fragment processing. PLoS Genet. 11, e1005659 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Davies, A. A., Huttner, D., Daigaku, Y., Chen, S. & Ulrich, H. D. Activation of ubiquitin-dependent DNA damage bypass is mediated by replication protein A. Mol. Cell 29, 625–636 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Watanabe, K. et al. Rad18 guides polη to replication stalling sites through physical interaction and PCNA monoubiquitination. EMBO J. 23, 3886–3896 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Toledo, L., Neelsen, K. J. & Lukas, J. Replication catastrophe: when a checkpoint fails because of exhaustion. Mol. Cell 66, 735–749 (2017).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  43. Guilliam, T. A. et al. Molecular basis for PrimPol recruitment to replication forks by RPA. Nat. Commun. 8, 15222 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Guilliam, T. A. & Doherty, A. J. PrimPol—prime time to reprime. Genes 8, 20 (2017).

    Article  PubMed Central  CAS  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Georgescu, R. et al. Structure of eukaryotic CMG helicase at a replication fork and implications to replisome architecture and origin initiation. Proc. Natl Acad. Sci. USA 114, E697–E706 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Zhou, J. C. et al. CMG–Pol epsilon dynamics suggests a mechanism for the establishment of leading-strand synthesis in the eukaryotic replisome. Proc. Natl Acad. Sci. USA 114, 4141–4146 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Ho, B., Baryshnikova, A. & Brown, G. W. Unification of protein abundance datasets yields a quantitative Saccharomyces cerevisiae proteome. Cell Syst. 6, 192–205 (2018).

    Article  CAS  PubMed  Google Scholar 

  49. Hirota, K. et al. In vivo evidence for translesion synthesis by the replicative DNA polymerase δ. Nucleic Acids Res. 44, 7242–7250 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Hirota, K. et al. The POLD3 subunit of DNA polymerase δ can promote translesion synthesis independently of DNA polymerase ζ. Nucleic Acids Res. 43, 1671–1683 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Tellier-Lebegue, C. et al. The translesion DNA polymerases Pol ζ and Rev1 are activated independently of PCNA ubiquitination upon UV radiation in mutants of DNA polymerase δ. PLoS Genet. 13, e1007119 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  52. Pagès, V., Maria, S. R. S., Prakash, L. & Prakash, S. Role of DNA damage-induced replication checkpoint in promoting lesion bypass by translesion synthesis in yeast. Genes Dev. 23, 1438–1449 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  53. Sabbioneda, S., Bortolomai, I., Giannattasio, M., Plevani, P. & Muzi-Falconi, M. Yeast Rev1 is cell cycle regulated, phosphorylated in response to DNA damage and its binding to chromosomes is dependent upon MEC1. DNA Repair 6, 121–127 (2007).

    Article  CAS  PubMed  Google Scholar 

  54. Zhao, L. & Washington, M. T. Translesion synthesis: insights into the selection and switching of DNA polymerases. Genes 8, 24 (2017).

    Article  PubMed Central  CAS  Google Scholar 

  55. Gallego-Sánchez, A., Andrés, S., Conde, F., San-Segundo, P. A. & Bueno, A. Reversal of PCNA ubiquitylation by Ubp10 in Saccharomyces cerevisiae. PLoS Genet. 8, e1002826 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  56. Kubota, T., Katou, Y., Nakato, R., Shirahige, K. & Donaldson, A. D. Replication-coupled PCNA unloading by the Elg1 complex occurs genome-wide and requires Okazaki fragment ligation. Cell Rep. 12, 774–787 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Zhuang, Z. et al. Regulation of polymerase exchange between Polη and Polδ by monoubiquitination of PCNA and the movement of DNA polymerase holoenzyme. Proc. Natl Acad. Sci. USA 105, 5361–5366 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Washington, M. T., Johnson, R. E., Prakash, S. & Prakash, L. Fidelity and processivity of Saccharomyces cerevisiae DNA polymerase η. J. Biol. Chem. 274, 36835–36838 (1999).

    Article  CAS  PubMed  Google Scholar 

  59. Sharp, P. M. & Li, W.-H. The codon adaptation index—a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res. 15, 1281–1295 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Frigola, J., Remus, D., Mehanna, A. & Diffley, J. F. X. ATPase-dependent quality control of DNA replication origin licensing. Nature 495, 339–343 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Coster, G., Frigola, J., Beuron, F., Morris, E. P. & Diffley, J. F. X. Origin licensing requires ATP binding and hydrolysis by the MCM replicative helicase. Mol. Cell 55, 666–677 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank J. Diffley for plasmids and yeast strains and J. Sale for critical reading of the manuscript. This work was supported by the Medical Research Council, as part of United Kingdom Research and Innovation (MRC grant no. MC_UP_1201/12 to J.T.P.Y). T.A.G. is supported by a Sir Henry Wellcome Postdoctoral Fellowship from the Wellcome Trust (213596/Z/18/Z).

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  1. Division of Protein and Nucleic Acid Chemistry, Medical Research Council Laboratory of Molecular Biology, Cambridge, UK

    Thomas A. Guilliam & Joseph T. P. Yeeles

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T.A.G. performed the experiments. T.A.G. and J.T.P.Y. wrote the manuscript.

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Correspondence to Joseph T. P. Yeeles.

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Extended data

Extended Data Fig. 1 Pol η promotes TLS of lagging and leading-strand CPDs.

a, Purified Okazaki fragment processing and TLS proteins. b, Long exposure of the denaturing gel shown in Fig. 1b showing the diffuse ~1.7 kb stall product produced on the lagging-strand CPD template. c, Two-dimensional gel of the reaction performed in the absence of Pol η on the undamaged leading-strand template, shown in lane 1 of main text Fig. 1d. d, Two-dimensional gel of the reaction performed in the absence of Pol η on the leading-strand CPD template, shown in lane 7 of main text Fig. 1d. e, Two-dimensional gel of the reaction performed in the presence of 16 nM Pol η on the leading-strand CPD template, shown in lane 12 of main text Fig. 1d.

Extended Data Fig. 2 Leading-strand TLS occurs uncoupled from CMG.

a, Oligonucleotide competition assay performed in the absence or presence of Pol η. Reaction products were cleaved with SwaI to truncate stall products before resolution on a urea polyacrylamide gel. Addition of Pol η promotes extension of the stall product in the gap left behind from oligonucleotide-mediated recoupling. b, Reaction scheme for the pulse-chase experiment shown in (c). c, Pulse chase experiment on the leading-strand CPD template with 5 nM Pol η added 3 min into the pulse, at the start of the chase, or 10 min into the chase.

Extended Data Fig. 3 Pol δ, but not Pol ε, inhibits lagging and leading-strand TLS.

a, Pol δ titration into standard replication reactions on the undamaged leading-strand template containing Fen1 and Ligase. b, Denaturing gel of the reaction products from main text Fig. 3a. c, Standard replication reaction on the lagging-strand CPD template in the presence of 5 nM Pol η and increasing concentrations of Pol δ, as performed in Fig. 3a, but in the absence of Fen1 and Ligase. d, Pol δ titration into standard replication reactions on the undamaged leading-strand template. e, Reaction scheme for the pulse-chase experiment shown in (f). f, Pulse chase experiment on the leading-strand CPD template with 5 nM Pol η alone, or with 5 nM extra Pol ε, or Pol δ, added at the start of the chase.

Extended Data Fig. 4 Uncoupled replication forks display a recoupling defect in the absence of Pol δ.

a, Reaction scheme for the pulse-chase experiment shown in (b). b, Pulse chase experiment on the leading-strand CPD template in the absence of Pol δ and the absence or presence of 5 nM Pol η, added at the start of the chase. c, Two-dimensional gel of the 20 min time point shown in lane 6 of (b). d, Two-dimensional gel of the 20 min time point shown in lane 12 of (b).

Extended Data Fig. 5 PCNA monoubiquitination stimulates on the fly TLS.

a, Western blot of PCNA from standard 60 min replication reactions on the leading-strand CPD template, or undamaged equivalent, in the absence or presence of Fen1 and Ligase. All reactions contained ubiquitin, Uba1, and Rad6–Rad18 in addition to standard replication proteins. Denaturing gel of reaction products is shown below. b, Standard replication reaction time course on the undamaged template performed in the absence or presence of Rad6–Rad18, Uba1, and ubiquitin. c, Standard replication reaction time course on the leading-strand CPD template in the presence of 2.5 nM Pol η and 0.3 nM or 2.5 nM Pol δ. Samples were treated with BamHI and SwaI to generate bypass and stall products prior to resolution on the urea polyacrylamide gel. d, Denaturing gel of the reaction products from Fig. 5c. e, Replication reaction time course performed on the leading-strand CPD template in the absence or presence of Uba1 or Rad6–Rad18. Reactions contained 2.5 nM Pol η, 2.5 nM Pol δ, 1 μM ubiquitin, 5 nM Fen1, and 5 nM Ligase, in addition to standard replication proteins. Urea polyacrylamide gel samples were treated with BamHI and SwaI to generate quantifiable bypass and stall products. f, Quantification of the data in (e) showing the percentage of bypass in the absence or presence of uba1 or Rad6–18. g, Replication reaction time course performed on the leading-strand CPD template in the presence of PCNA monoubiquitination machinery (Rad6–Rad18, Uba1, and ubiquitin) and Pol η and the absence or presence of Pol δ.

Extended Data Fig. 6 Characterization of PCNAK164R.

a, Standard replication reaction time course performed on the undamaged template with either wild type PCNA or PCNAK164R. b, Standard replication reactions on the leading-strand CPD template containing increasing amounts of wild type PCNA or PCNAK164R. Reactions contained 5 nM Pol η. c, Standard replication reactions on the leading-strand CPD template in the absence or presence of 2.5 nM Pol δ and increasing amounts of wild type PCNA. Reactions contained 5 nM Pol η. d, Standard replication reaction time course on the undamaged leading-strand template with either wild type PCNA or PCNAK164R in the presence of Fen1 and Ligase.

Extended Data Fig. 7 PCNA monoubiquitination stimulates on the fly TLS in the absence of Fen1 and Ligase.

a, Standard replication reaction time course performed with wild-type PCNA in the absence or presence of Uba1. Reactions contained 10 nM Pol η, 10 nM Pol δ, 250 nM ubiquitin, and 200 nM Rad6–Rad18, in addition to standard replication proteins. Urea polyacrylamide gel samples were treated with BamHI and SwaI to generate quantifiable bypass and stall products. b, Quantification of the percentage of bypass in the absence or presence of Uba1 as performed in (b). Data are plotted as the means and s.e.m. of three independent experiments. c, Standard replication reaction time course performed with PCNAK164R in the absence or presence of Uba1. Reactions contained 10 nM Pol η, 10 nM Pol δ, 250 nM ubiquitin, and 200 nM Rad6–Rad18, in addition to standard replication proteins. Urea polyacrylamide gel samples were treated with BamHI and SwaI to generate quantifiable bypass and stall products. d, Quantification of the percentage of bypass in the absence or presence of Uba1 as performed in (d). Data are plotted as the means and s.e.m. of three independent experiments.

Extended Data Fig. 8 PCNA monoubiquitination promotes lagging-strand TLS.

a, Standard replication reaction time course on the lagging-strand CPD template in the presence of 2.5 nM Pol η and 0.3125 nM or 2.5 nM Pol δ. Samples were treated with BamHI and SwaI to generate bypass and stall products prior to resolution on the urea polyacrylamide gel. b, Denaturing gel of the reaction products from Fig. 6a.

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Guilliam, T.A., Yeeles, J.T.P. Reconstitution of translesion synthesis reveals a mechanism of eukaryotic DNA replication restart. Nat Struct Mol Biol 27, 450–460 (2020). https://doi.org/10.1038/s41594-020-0418-4

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