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.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Nature Communications Open Access 01 April 2022
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
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.
Macheret, M. & Halazonetis, T. D. DNA replication stress as a hallmark of cancer. Annu. Rev. Pathol. 10, 425–448 (2015).
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).
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).
McIntosh, D. & Blow, J. J. Dormant origins, the licensing checkpoint, and the response to replicative stresses. Cold Spring Harb. Perspect. Biol. 4, a012955 (2012).
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).
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).
Hyrien, O. Peaks cloaked in the mist: The landscape of mammalian replication origins. J. Cell Biol. 208, 147–160 (2015).
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).
Smith, D. J., Yadav, T. & Whitehouse, I. Detection and sequencing of Okazaki fragments in S. cerevisiae. DNA Replication 1300, 141–153 (2015).
Smith, D. J. & Whitehouse, I. Intrinsic coupling of lagging-strand synthesis to chromatin assembly. Nature 483, 434–438 (2012).
Bielinsky, A.-K. G. & Susan, A. Chromosomal ARS1 has a single leading strand start site. Mol. Cell 3, 477–486 (1999).
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).
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).
Petryk, N. et al. Replication landscape of the human genome. Nat. Commun. 7, 10208 (2016).
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).
Tubbs, A. et al. Dual roles of poly(dA:dT) tracts in replication initiation and fork collapse. Cell 174, 1127–1142.e1119 (2018).
Chen, Y.-H. et al. Transcription shapes DNA replication initiation and termination in human cells. Nat. Struct. Mol. Biol. 26, 67–77 (2018).
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).
Meldal, M. & Tornøe, C. W. Cu-catalyzed azide-alkyne cycloaddition. Chem. Rev. 108, 2952–3015 (2008).
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).
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).
Gilbert, D. M. Evaluating genome-scale approaches to eukaryotic DNA replication. Nat. Rev. Genet. 11, 673–684 (2010).
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).
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).
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).
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).
Besnard, E. et al. Best practices for mapping replication origins in eukaryotic chromosomes. Curr. Protoc. Cell. Biol. 64, 22 18 21–13 (2014).
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).
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).
Bielinsky, A.-K. & Gerbi, S. A. Discrete start sites for DNA synthesis in the yeast ARS1 origin. Science 279, 95–98 (1998).
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).
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).
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).
Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).
Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).
Ramírez, F. et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 44, W160–W165 (2016).
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).
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).
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.
The authors declare no competing interests.
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.
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
About this article
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