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

Sarah Kit Leng Lui, Sarah Keegan, Peter Tonzi, Malik Kahli, Yu-Hung Chen, Noor Chalhoub, Kate E. Coleman, David Fenyo, Duncan J. Smith, Tony T. Huang

Published: 2021-01-12 DOI: 10.1038/s41596-020-00454-5

Okazaki fragment sequencing

genome-wide replication

DNA replication dynamics

mammalian cells

replication fork directionality

AI 解读

Author Correction: Full-length sequencing of circular DNA viruses and extrachromosomal circular DNA using CIDER-Seq

Generation of heart-forming organoids from human pluripotent stem cells

Design and fabrication of wearable electronic textiles using twisted fiber-based threads

Activation of mechanoluminescent nanotransducers by focused ultrasound enables light delivery to deep-seated tissue in vivo

Chromatin accessibility profiling by ATAC-seq

Deoxyfluorination of phenols for chemoselective18F-labeling of peptides

Author Correction: cfSNV: a software tool for the sensitive detection of somatic mutations from cell-free DNA

Human primordial germ cell-like cells specified from resetting precursors develop in human hindgut organoids

An inexpensive semi-automated sample processing pipeline for cell-free RNA extraction

Application of CHyMErA Cas9-Cas12a combinatorial genome-editing platform for genetic interaction mapping and gene fragment deletion screening

查看全部

Sections

Figures

References

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 protoco
Introduction
Materials
Procedure
Troubleshooting
Timing
Anticipated results
- References
- References
References
ACKNOWLEDGMENTS
ACKNOWLEDGMENTS

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).

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

推荐阅读