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.

Monitoring genome-wide replication fork directionality by Okazaki fragment sequencing in mammalian cells

Abstract

The ability to monitor DNA replication fork directionality at the genome-wide scale is paramount for a greater understanding of how genetic and environmental perturbations can impact replication dynamics in human cells. Here we describe a detailed protocol for isolating and sequencing Okazaki fragments from asynchronously growing mammalian cells, termed Okazaki fragment sequencing (Ok-seq), for the purpose of quantitatively determining replication initiation and termination frequencies around specific genomic loci by meta-analyses. Briefly, cells are pulsed with 5-ethynyl-2′-deoxyuridine (EdU) to label newly synthesized DNA, and collected for DNA extraction. After size fractionation on a sucrose gradient, Okazaki fragments are concentrated and purified before click chemistry is used to tag the EdU label with a biotin conjugate that is cleavable under mild conditions. Biotinylated Okazaki fragments are then captured on streptavidin beads and ligated to Illumina adapters before library preparation for Illumina sequencing. The use of Ok-seq to interrogate genome-wide replication fork initiation and termination efficiencies can be applied to all unperturbed, asynchronously growing mammalian cells or under conditions of replication stress, and the assay can be performed in less than 2 weeks.

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: Schematic overview of Ok-seq protocol.
Fig. 2: Schematic representation of expected Okazaki fragment distributions for replication initiation and termination sites.
Fig. 3: Anticipated results from RPE-1 cells.
Fig. 4: Agarose gel and TapeStation results.

Data availability

Data used for the non-downsampled (100%) curves in Fig. 4 are provided at GSE114017. The downsampled curves can be produced by repeatedly removing one-half of the reads from the fastq files and then re-running the processing pipeline on the reduced dataset.

Code availability

All code is publicly available under the GNU General Public License v2.0 in our GitHub repository: https://github.com/FenyoLab/Ok-Seq_Processing. Included in the repository is example input data for creating plots as shown in Fig. 4, as well as additional plots as described in the ‘Data analysis’ section. The expected output of all scripts is also included as pdf/jpg files. Documentation is provided as a readme file, and specific instructions on parameters to functions are embedded as inline comments in the code. The current version of the software is v1.0, tagged as a release in GitHub: https://github.com/FenyoLab/Ok-Seq_Processing/releases/tag/v1.0.

References

  1. 1.

    Macheret, M. & Halazonetis, T. D. DNA replication stress as a hallmark of cancer. Annu. Rev. Pathol. 10, 425–448 (2015).

    CAS  Article  Google Scholar 

  2. 2.

    Fragkos, M., Ganier, O., Coulombe, P. & Mechali, M. DNA replication origin activation in space and time. Nat. Rev. Mol. Cell Biol. 16, 360–374 (2015).

    CAS  Article  Google Scholar 

  3. 3.

    Ibarra, A., Schwob, E. & Méndez, J. Excess MCM proteins protect human cells from replicative stress by licensing backup origins of replication. Proc. Natl Acad. Sci. USA 105, 8956–8961 (2008).

    CAS  Article  Google Scholar 

  4. 4.

    McIntosh, D. & Blow, J. J. Dormant origins, the licensing checkpoint, and the response to replicative stresses. Cold Spring Harb. Perspect. Biol. 4, a012955 (2012).

    Article  Google Scholar 

  5. 5.

    Ge, X. Q., Jackson, D. A. & Blow, J. J. Dormant origins licensed by excess Mcm2-7 are required for human cells to survive replicative stress. Genes Dev. 21, 3331–3341 (2007).

    CAS  Article  Google Scholar 

  6. 6.

    Kawabata, T. et al. Stalled fork rescue via dormant replication origins in unchallenged S phase promotes proper chromosome segregation and tumor suppression. Mol. Cell 41, 543–553 (2011).

    CAS  Article  Google Scholar 

  7. 7.

    Hyrien, O. Peaks cloaked in the mist: The landscape of mammalian replication origins. J. Cell Biol. 208, 147–160 (2015).

    CAS  Article  Google Scholar 

  8. 8.

    Okazaki, R., Okazaki, T., Sakabe, K., Sugimoto, K. & Sugino, A. Mechanism of DNA chain growth, I. Possible discontinuity and unusual secondary structure of newly synthesized chains. Proc. Natl Acad. Sci. USA 59, 598–605 (1968).

    CAS  Article  Google Scholar 

  9. 9.

    Smith, D. J., Yadav, T. & Whitehouse, I. Detection and sequencing of Okazaki fragments in S. cerevisiae. DNA Replication 1300, 141–153 (2015).

    CAS  Article  Google Scholar 

  10. 10.

    Smith, D. J. & Whitehouse, I. Intrinsic coupling of lagging-strand synthesis to chromatin assembly. Nature 483, 434–438 (2012).

    CAS  Article  Google Scholar 

  11. 11.

    Bielinsky, A.-K. G. & Susan, A. Chromosomal ARS1 has a single leading strand start site. Mol. Cell 3, 477–486 (1999).

    CAS  Article  Google Scholar 

  12. 12.

    Pourkarimi, E., Bellush, J. M. & Whitehouse, I. Spatiotemporal coupling and decoupling of gene transcription with DNA replication origins during embryogenesis in C. elegans. eLife 5, e21728 (2016).

    Article  Google Scholar 

  13. 13.

    Sriramachandran, A. M. et al. Genome-wide nucleotide-resolution mapping of DNA replication patterns, single-strand breaks, and lesions by GLOE-Seq. Mol. Cell 78, 975–985 e977 (2020).

    CAS  Article  Google Scholar 

  14. 14.

    Petryk, N. et al. Replication landscape of the human genome. Nat. Commun. 7, 10208 (2016).

    CAS  Article  Google Scholar 

  15. 15.

    Osmundson, J. S., Kumar, J., Yeung, R. & Smith, D. J. Pif1-family helicases cooperatively suppress widespread replication-fork arrest at tRNA genes. Nat. Struct. Mol. Biol. 24, 162–170 (2017).

    CAS  Article  Google Scholar 

  16. 16.

    Tubbs, A. et al. Dual roles of poly(dA:dT) tracts in replication initiation and fork collapse. Cell 174, 1127–1142.e1119 (2018).

    CAS  Article  Google Scholar 

  17. 17.

    Chen, Y.-H. et al. Transcription shapes DNA replication initiation and termination in human cells. Nat. Struct. Mol. Biol. 26, 67–77 (2018).

    Article  Google Scholar 

  18. 18.

    Kahli, M., Osmundson, J. S., Yeung, R. & Smith, D. J. Processing of eukaryotic Okazaki fragments by redundant nucleases can be uncoupled from ongoing DNA replication in vivo. Nucleic Acids Res. 47, 1814–1822 (2019).

    CAS  Article  Google Scholar 

  19. 19.

    Meldal, M. & Tornøe, C. W. Cu-catalyzed azide-alkyne cycloaddition. Chem. Rev. 108, 2952–3015 (2008).

    CAS  Article  Google Scholar 

  20. 20.

    Salic, A. & Mitchison, T. J. A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc. Natl Acad. Sci. USA 105, 2415–2420 (2008).

    CAS  Article  Google Scholar 

  21. 21.

    Koç, A., Wheeler, L. J., Mathews, C. K. & Merrill, G. F. Hydroxyurea arrests DNA replication by a mechanism that preserves basal dNTP pools. J. Biol. Chem. 279, 223–230 (2004).

    Article  Google Scholar 

  22. 22.

    Gilbert, D. M. Evaluating genome-scale approaches to eukaryotic DNA replication. Nat. Rev. Genet. 11, 673–684 (2010).

    CAS  Article  Google Scholar 

  23. 23.

    Mesner, L. D. et al. Bubble-seq analysis of the human genome reveals distinct chromatin-mediated mechanisms for regulating early- and late-firing origins. Genome Res. 23, 1774–1788 (2013).

    CAS  Article  Google Scholar 

  24. 24.

    Langley, A. R., Gräf, S., Smith, J. C. & Krude, T. Genome-wide identification and characterisation of human DNA replication origins by initiation site sequencing (ini-seq). Nucleic Acids Res. 44, 10230–10247 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Macheret, M. & Halazonetis, T. D. Monitoring early S-phase origin firing and replication fork movement by sequencing nascent DNA from synchronized cells. Nat. Protoc. 14, 51–67 (2018).

    Article  Google Scholar 

  26. 26.

    Giacca, M., Pelizon, C. & Falaschi, A. Mapping replication origins by quantifying relative abundance of nascent DNA strands using competitive polymerase chain reaction. Methods Enzymol. 13, 301–312 (1997).

    CAS  Article  Google Scholar 

  27. 27.

    Besnard, E. et al. Best practices for mapping replication origins in eukaryotic chromosomes. Curr. Protoc. Cell. Biol. 64, 22 18 21–13 (2014).

    Google Scholar 

  28. 28.

    Zhao, P. A., Sasaki, T. & Gilbert, D. M. High-resolution Repli-Seq defines the temporal choreography of initiation, elongation and termination of replication in mammalian cells. Genome Biol. 21, 76 (2020).

    CAS  Article  Google Scholar 

  29. 29.

    Vassilev, L. & Johnson, E. M. Mapping initiation sites of DNA replication in vivo using polymerase chain reaction amplification of nascent strand segments. Nucleic Acids Res. 17, 7693–7705 (1989).

    CAS  Article  Google Scholar 

  30. 30.

    Bielinsky, A.-K. & Gerbi, S. A. Discrete start sites for DNA synthesis in the yeast ARS1 origin. Science 279, 95–98 (1998).

    CAS  Article  Google Scholar 

  31. 31.

    Foulk, M. S., Urban, J. M., Casella, C. & Gerbi, S. A. Characterizing and controlling intrinsic biases of lambda exonuclease in nascent strand sequencing reveals phasing between nucleosomes and G-quadruplex motifs around a subset of human replication origins. Genome Res. 25, 725–735 (2015).

    CAS  Article  Google Scholar 

  32. 32.

    Chen, Y. H. et al. ATR-mediated phosphorylation of FANCI regulates dormant origin firing in response to replication stress. Mol. Cell 58, 323–338 (2015).

    CAS  Article  Google Scholar 

  33. 33.

    Wu, X. et al. Developmental and cancer-associated plasticity of DNA replication preferentially targets GC-poor, lowly expressed and late-replicating regions. Nucleic Acids Res. 46, 10157–10172 (2018).

    CAS  Article  Google Scholar 

  34. 34.

    Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    CAS  Article  Google Scholar 

  35. 35.

    Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  Google Scholar 

  36. 36.

    Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    CAS  Article  Google Scholar 

  37. 37.

    Ramírez, F. et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 44, W160–W165 (2016).

    Article  Google Scholar 

  38. 38.

    Ligasova, A. et al. Dr Jekyll and Mr Hyde: a strange case of 5-ethynyl-2′-deoxyuridine and 5-ethynyl-2′-deoxycytidine. Open Biol. 6, 150172 (2016).

    Article  Google Scholar 

  39. 39.

    Manska, S., Octaviano, R. & Rossetto, C. C. 5-Ethynyl-2'-deoxycytidine and 5-ethynyl-2'-deoxyuridine are differentially incorporated in cells infected with HSV-1, HCMV, and KSHV viruses. J. Biol. Chem. 295, 5871–5890 (2020).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

The authors thank A. Heguy and O. Zappile from the NYU Genome Technology Center for assistance with TapeStation and sequencing. Work in the T.T.H. laboratory is supported by grants from the NIH (ES025166), V Foundation BRCA Research and Basser Innovation Award. Work in D.J.S. laboratory is supported by grant (R35 GM134918) from the NIH.

Author information

Affiliations

Authors

Contributions

Y.-H.C. and M.K. developed the original protocol from our labs, P.T., S.K.L.L., N.C., and K.E.C. made modifications in the DNA extraction steps and performed the experiments, and S.K., D.F., and D.J.S. provided bioinformatics support. S.K.L.L. and S.K. wrote the manuscript, S.K., D.F., D.J.S., and T.T.H. analyzed the data, and T.T.H. and D.J.S. conceived the project.

Corresponding authors

Correspondence to David Fenyo or Duncan J. Smith or Tony T. Huang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Anja-Katrin Bielinsky, Conrad A. Nieduszynski, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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

Related links

Key reference using this protocol

Chen, Y. et al. Nat. Struct. Mol. Biol. 26, 67–77 (2019): https://doi.org/10.1038/s41594-018-0171-0

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kit Leng Lui, S., Keegan, S., Tonzi, P. et al. Monitoring genome-wide replication fork directionality by Okazaki fragment sequencing in mammalian cells. Nat Protoc 16, 1193–1218 (2021). https://doi.org/10.1038/s41596-020-00454-5

Download citation

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