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.

Fast and efficient DNA replication with purified human proteins

Abstract

Chromosome replication is performed by a complex and intricate ensemble of proteins termed the replisome, where the DNA polymerases Polδ and Polε, DNA polymerase α-primase (Polα) and accessory proteins including AND-1, CLASPIN and TIMELESS–TIPIN (respectively known as Ctf4, Mrc1 and Tof1–Csm3 in Saccharomyces cerevisiae) are organized around the CDC45–MCM–GINS (CMG) replicative helicase1,2,3,4,5,6,7. Because a functional human replisome has not been reconstituted from purified proteins, how these factors contribute to human DNA replication and whether additional proteins are required for optimal DNA synthesis are poorly understood. Here we report the biochemical reconstitution of human replisomes that perform fast and efficient DNA replication using 11 purified human replication factors made from 43 polypeptides. Polε, but not Polδ, is crucial for optimal leading-strand synthesis. Unexpectedly, Polε-mediated leading-strand replication is highly dependent on the sliding-clamp processivity factor PCNA and the alternative clamp loader complex CTF18–RFC. We show how CLASPIN and TIMELESS–TIPIN contribute to replisome progression and demonstrate that, in contrast to the budding yeast replisome8, AND-1 directly augments leading-strand replication. Moreover, although AND-1 binds to Polα9,10, the interaction is dispensable for lagging-strand replication, indicating that Polα is functionally recruited via an AND-1-independent mechanism for priming in the human replisome. Collectively, our work reveals how the human replisome achieves fast and efficient leading-strand and lagging-strand DNA replication, and provides a powerful system for future studies of the human replisome and its interactions with other DNA metabolic processes.

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: PCNA is required for efficient leading-strand synthesis by CMG–Polε.
Fig. 2: Reconstitution of cellular DNA replication rates with purified proteins.
Fig. 3: PCNA loading by CTF18–RFC coupled to Polε is required for maximal replication rates.
Fig. 4: How TIM–TIPIN, CLASPIN and AND-1 facilitate leading-strand replication.
Fig. 5: Reconstitution of lagging-strand DNA replication with purified human proteins.

Data availability

The data supporting the findings of this study are available within the paper and its Supplementary Information files.

References

  1. Baretić, D. et al. Cryo-EM structure of the fork protection complex bound to CMG at a replication fork. Mol. Cell 78, 926–940.e13 (2020).

    Article  Google Scholar 

  2. Jones, M. L., Baris, Y., Taylor, M. R. G. & Yeeles, J. T. P. Structure of a human replisome shows the organisation and interactions of a DNA replication machine. EMBO J. 40, e108819 (2021).

    CAS  Article  Google Scholar 

  3. 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).

    ADS  Article  Google Scholar 

  4. Yuan, Z. et al. Ctf4 organizes sister replisomes and Pol alpha into a replication factory. eLife 8, e47405 (2019).

    CAS  Article  Google Scholar 

  5. Rzechorzek, N. J. et al. CryoEM structures of human CMG–ATPγS–DNA and CMG–AND-1 complexes. Nucleic Acids Res. 48, 6980–6995 (2020).

    CAS  Article  Google Scholar 

  6. Kapadia, N. et al. Processive activity of replicative DNA polymerases in the replisome of live eukaryotic cells. Mol. Cell 80, 114–126.e8 (2020).

    CAS  Article  Google Scholar 

  7. Lewis, J. S. et al. Tunability of DNA polymerase stability during eukaryotic DNA replication. Mol. Cell 77, 17–25.e5 (2020).

    CAS  Article  Google Scholar 

  8. 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  Article  Google Scholar 

  9. Kilkenny, M. L. et al. The human CTF4-orthologue AND-1 interacts with DNA polymerase alpha/primase via its unique C-terminal HMG box. Open Biol. 7, 170217 (2017).

    Article  Google Scholar 

  10. Guan, C., Li, J., Sun, D., Liu, Y. & Liang, H. The structure and polymerase-recognition mechanism of the crucial adaptor protein AND-1 in the human replisome. J. Biol. Chem. 292, 9627–9636 (2017).

    CAS  Article  Google Scholar 

  11. Petermann, E., Helleday, T. & Caldecott, K. W. Claspin promotes normal replication fork rates in human cells. Mol. Biol. Cell 19, 2373–2378 (2008).

    CAS  Article  Google Scholar 

  12. Conti, C. et al. Replication fork velocities at adjacent replication origins are coordinately modified during DNA replication in human cells. Mol. Biol. Cell 18, 3059–3067 (2007).

    CAS  Article  Google Scholar 

  13. Somyajit, K. et al. Redox-sensitive alteration of replisome architecture safeguards genome integrity. Science 358, 797–802 (2017).

    ADS  CAS  Article  Google Scholar 

  14. Abe, T. et al. AND-1 fork protection function prevents fork resection and is essential for proliferation. Nat. Commun. 9, 3091 (2018).

    ADS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

  17. Aria, V. & Yeeles, J. T. P. Mechanism of bidirectional leading-strand synthesis establishment at eukaryotic DNA replication origins. Mol. Cell 73, 199–211.e10 (2019).

    CAS  Article  Google Scholar 

  18. Grabarczyk, D. B., Silkenat, S. & Kisker, C. Structural basis for the recruitment of Ctf18-RFC to the replisome. Structure 26, 137–144.e3 (2018).

    CAS  Article  Google Scholar 

  19. Stokes, K., Winczura, A., Song, B., Piccoli, G. & Grabarczyk, D. B. Ctf18-RFC and DNA Pol form a stable leading strand polymerase/clamp loader complex required for normal and perturbed DNA replication. Nucleic Acids Res. 48, 8128–8145 (2020).

    CAS  Article  Google Scholar 

  20. Murakami, T. et al. Stable interaction between the human proliferating cell nuclear antigen loader complex Ctf18-replication factor C (RFC) and DNA polymerase ε is mediated by the cohesion-specific subunits, Ctf18, Dcc1, and Ctf8*. J. Biol. Chem. 285, 34608–34615 (2010).

    CAS  Article  Google Scholar 

  21. Fujisawa, R., Ohashi, E., Hirota, K. & Tsurimoto, T. Human CTF18-RFC clamp-loader complexed with non-synthesising DNA polymerase ε efficiently loads the PCNA sliding clamp. Nucleic Acids Res. 45, 4550–4563 (2017).

    CAS  Article  Google Scholar 

  22. Tunyasuvunakool, K. et al. Highly accurate protein structure prediction for the human proteome. Nature 596, 590–596 (2021).

    ADS  CAS  Article  Google Scholar 

  23. 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  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  25. Terret, M. E., Sherwood, R., Rahman, S., Qin, J. & Jallepalli, P. V. Cohesin acetylation speeds the replication fork. Nature 462, 231–234 (2009).

    ADS  CAS  Article  Google Scholar 

  26. Crabbe, L. et al. Analysis of replication profiles reveals key role of RFC-Ctf18 in yeast replication stress response. Nat. Struct. Mol. Biol. 17, 1391–1397 (2010).

    CAS  Article  Google Scholar 

  27. Hanna, J. S., Kroll, E. S., Lundblad, V. & Spencer, F. A. Saccharomyces cerevisiae CTF18 and CTF4 are required for sister chromatid cohesion. Mol. Cell. Biol. 21, 3144–3158 (2001).

    CAS  Article  Google Scholar 

  28. Mayer, M. L., Gygi, S. P., Aebersold, R. & Hieter, P. Identification of RFC(Ctf18p, Ctf8p, Dcc1p): an alternative RFC complex required for sister chromatid cohesion in S. cerevisiae. Mol. Cell 7, 959–970 (2001).

    CAS  Article  Google Scholar 

  29. Kawasumi, R. et al. Vertebrate CTF18 and DDX11 essential function in cohesion is bypassed by preventing WAPL-mediated cohesin release. Genes Dev. 35, 1368–1382 (2021).

    CAS  Article  Google Scholar 

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

    Article  Google Scholar 

  31. Henricksen, L. A., Umbricht, C. B. & Wold, M. S. Recombinant replication protein A: expression, complex formation, and functional characterization. J. Biol. Chem. 269, 11121–11132 (1994).

    CAS  Article  Google Scholar 

  32. Sebesta, M. et al. Role of PCNA and TLS polymerases in D-loop extension during homologous recombination in humans. DNA Repair 12, 691–698 (2013).

    CAS  Article  Google Scholar 

  33. Xing, X. et al. A recurrent cancer-associated substitution in DNA polymerase ε produces a hyperactive enzyme. Nat. Commun. 10, 374 (2019).

    ADS  CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank J. Shi for operation of the LMB baculovirus facility, L. Passmore for protein expression vectors, M. Jones for advice on glycerol gradients, and L. Krejci and M. Wold for the RPA expression plasmid. This work was supported by the MRC, as part of UK Research and Innovation (MRC grant MC_UP_1201/12 to J.T.P.Y.), the Wellcome Trust (reference 110014/Z/15/Z) for a Sir Henry Wellcome Postdoctoral Fellowship to M.R.G.T and an LMB Cambridge Trust International Scholarship to Y.B.

Author information

Authors and Affiliations

Authors

Contributions

Y.B. performed all experiments, generated the expression vectors, prepared DNA templates and purified proteins, wrote the methods, and reviewed and edited the manuscript. M.R.G.T. conceptualized the study, acquired funding, performed preliminary DNA replication assays, generated the expression vectors, prepared DNA templates and purified proteins, and reviewed the manuscript. V.A. identified the CMG–Polα interaction. J.T.P.Y. conceptualized and supervised the study, acquired funding, prepared the DNA template and performed protein purification, wrote the original draft of the manuscript, and reviewed and edited the manuscript.

Corresponding author

Correspondence to Joseph T. P. Yeeles.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature thanks David Gilbert, Bruce Stillman and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. 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 figures and tables

Extended Data Fig. 1 Purified human DNA replication proteins.

Coomassie stained SDS-PAGE of human DNA replication proteins. Individual lanes from the gel in Fig. 1a are shown with each subunit labelled.

Extended Data Fig. 2 Leading-strand synthesis.

a, Standard replication reaction on the 9.7 kbp template performed with the indicated proteins and analysed by native and denaturing agarose gel electrophoresis as indicated. In the absence of RFC and PCNA the predominant replication products (intermediates) migrate above the position of full length in the native gel. As indicated, in the native gel template labelling products and complete full-length replication products migrate in the same position. b, Denaturing agarose gel analysis of a pulse chase experiment on the 9.7 kbp template with the indicated proteins. Unless otherwise stated, in this and all pulse chase experiments, the chase was added at 50 s. c, Denaturing agarose gel analysis of a replication reaction on the 9.7 kbp template with the indicated proteins. d, Denaturing agarose gel analysis of a pulse chase experiment on the 9.7 kbp template with the indicated proteins.

Extended Data Fig. 3 CTF18-RFC is required for optimal leading-strand synthesis.

a, Denaturing agarose gel analysis of a replication reaction on the 9.7 kbp template with the indicated proteins. b, Denaturing agarose gel analysis of a pulse chase experiment on the 9.7 kbp template with the indicated proteins. c, Silver-stained SDS-PAGE analysis of glycerol gradients performed with the indicated proteins. For clarity, only CMG, CTF18-RFC and Pol ε subunits are annotated. d, Coomassie stained SDS-PAGE of CTF18-RFC and Pol ε interaction mutants. e, Silver-stained SDS-PAGE analysis of a pull-down experiment with the indicated proteins showing that mutation of CTF18 and POLE1 disrupt the interaction between the two proteins. f, Denaturing agarose gel analysis of a replication reaction performed for 3 min on the 9.7 kbp template with the indicated proteins.

Extended Data Fig. 4 CTF18-RFC accelerates established replication forks.

a, Lane profiles of the 165 s timepoints in Fig. 3e where CTF18-RFC was absent or added in the chase (lanes 2 and 8 respectively). b, Denaturing agarose gel analysis of a pulse chase experiment on the 15.8 kbp template with the indicated proteins. Where indicated CTF18-RFC or CTF18-RFCRAA were added with the chase. c, Lane profiles of the 240 s timepoints in (b). For lane profiles (a, c), product intensities were normalised by dividing each value by the relative intensity of the total signal in a given lane.

Extended Data Fig. 5 PCNA loading by CTF18-RFC is required for optimal leading-strand synthesis.

a, Coomassie stained SDS-PAGE of CTF18-1-8 module complexes. b, Silver-stained SDS-PAGE analysis of a pull-down experiment with the indicated proteins showing that the CTF18-1-8 module interacts specifically with Pol ε. c, Coomassie stained SDS-PAGE of WT and CTF18K380E complexes. d, Silver-stained SDS-PAGE analysis of a pull-down experiment with the indicated proteins showing that CTF18K380E-RFC retains the capacity to interact with Pol ε. e, f, Primer extension reactions on M13mp18 single-strand DNA with Pol δ and Pol ε showing that CTF18K380E-RFC has a severe defect in supporting PCNA-dependent DNA synthesis by both polymerases. g, (left) Denaturing agarose gel analysis of a pulse-chase experiment on the 15.8 kbp template with the indicated proteins. The chase was added at 1 min 45 s. Where indicated CTF18-RFC or CTF18K380E-RFC were added with the chase. (right) Lane profiles for the 5 min timepoint. Data were normalised by dividing each intensity value by the relative total signal at the 2 min timepoint.

Extended Data Fig. 6 TIM-TIPIN, CLASPIN and AND-1 enhance leading-strand replication.

a, Denaturing agarose gel analysis of a time course experiment on the 15.8 kbp template with the indicated proteins at two concentrations of potassium glutamate (K-Glu). b, c, Lane profiles of the data in Fig. 4a, b respectively. d, Denaturing agarose gel analysis (top) and lane profiles (bottom) of a 3 min 45 s replication reaction on the 15.8 kbp template with the indicated proteins. e, Lane profiles of the data in Fig. 4c. TT, TIM-TIPIN. f, Denaturing agarose gel analysis (top) and lane profile (bottom) of a 3 min 45 s replication reaction on the 15.8 kbp template with the indicated proteins. TT, TIM-TIPIN. g, Denaturing agarose gel analysis of a 3.5 min replication reaction on the 15.8 kbp template with the indicated proteins. In d, f, g, the potassium glutamate concentration was 250 mM. For lane profiles (bf), product intensities were normalised by dividing each value by the relative intensity of the total signal in a given lane.

Extended Data Fig. 7 CLASPIN and AND-1 truncations.

a, Cartoon representation of the core human replisome (PDB:7PFO)2 showing the region of CLASPIN (E284–K319) that interacts with the TIM α-solenoid. b, Coomassie stained SDS-PAGE of CLASPIN truncation mutants. c, d Lane profiles of the data in Fig. 4e. e, Coomassie stained SDS-PAGE of AND-1 truncation mutants. f, Lane profiles of the data in Fig. 4g. For lane profiles (c, d, f), product intensities were normalised by dividing each value by the relative intensity of the total signal in a given lane.

Extended Data Fig. 8 Reconstitution of lagging-strand replication.

a, Denaturing agarose gel analysis of a 20 min replication on the 9.7 kbp template with the indicated proteins. b, Lane profiles from lanes 3 and 4 in (a). c, Schematic showing the possible replication products (–/+ Pol δ) if lagging strands are extended by Pol δ and are constituents of both replication intermediates and full-length products. d, e, Two-dimensional agarose gel analysis of 20 min replication reactions performed with the indicated proteins on the 9.7 kbp template in the absence (d) and presence (e) of Pol δ. In all reactions, the concentration of potassium glutamate was 250 mM.

Extended Data Fig. 9 Lagging-strand replication occurs at all replication forks.

a, Schematic of a replication reaction on the cyclobutane pyrimidine dimer (CPD) template. b, Native and denaturing gel analysis of a time course experiment on undamaged and CPD templates with the indicated proteins. c, Native and denaturing gel analysis of a 60 min reaction on the CPD template with different concentrations of Pol α as indicated. d, e, Two-dimensional agarose gel analysis of 30 min replication reactions performed with the indicated proteins on the CPD template in the absence (d) and presence (e) of Pol α. In all reactions, the concentration of potassium glutamate was 250 mM.

Extended Data Fig. 10 Role of AND-1 in lagging-strand replication.

a, Lane profiles from Fig. 5d, lanes 5 and 6. b, Lane profiles from the experiment in Fig. 5e, lanes 2, 3, 5, 7, 8 and 10. c, Denaturing agarose gel analysis of a 30 min reaction on the 9.7 kbp template with the indicated proteins. d, Silver-stained SDS-PAGE analysis of glycerol gradients performed with the indicated proteins demonstrating complex formation between Pol α and CMG in the absence of replication fork DNA.

Supplementary information

Supplementary

This file contains Supplementary Tables 1–2 and Supplementary Fig. 1 (the uncropped gels).

Reporting Summary

Peer Review File

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Baris, Y., Taylor, M.R.G., Aria, V. et al. Fast and efficient DNA replication with purified human proteins. Nature 606, 204–210 (2022). https://doi.org/10.1038/s41586-022-04759-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-022-04759-1

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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