Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing

Methods : a Companion to Methods in Enzymology
Julien N BiancoPhilippe Pasero

Abstract

DNA combing is a powerful method developed by Bensimon and colleagues to stretch DNA molecules on silanized glass coverslips. This technique provides a unique way to monitor the activation of replication origins and the progression of replication forks at the level of single DNA molecules, after incorporation of thymidine analogs, such as 5-bromo-2'-deoxyuridine (BrdU), 5-iodo-2'-deoxyuridine (IdU) and 5-chloro-2'-deoxyuridine (CldU) in newly-synthesized DNA. Unlike microarray-based approaches, this assay gives access to the variability of replication profiles in individual cells. It can also be used to monitor the effect of DNA lesions on fork progression, arrest and restart. In this review, we propose standard DNA combing methods to analyze DNA replication in budding yeast and in human cells. We also show that 5-ethynyl-2'-deoxyuridine (EdU) can be used as a good alternative to BrdU for DNA combing analysis, as unlike halogenated nucleotides, it can be detected without prior denaturation of DNA.

References

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Sep 22, 2009·Current Opinion in Cell Biology·Dirk Remus, John F X Diffley

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Citations

Apr 18, 2014·Proceedings of the National Academy of Sciences of the United States of America·Yi-Jun SheuBruce Stillman
Jan 1, 2014·The Journal of Cell Biology·Jakob MejlvangAnja Groth
Jan 20, 2016·Scientific Reports·Atanas KaykovPaul Nurse
Oct 1, 2013·Journal of Molecular Biology·Kazumasa YoshidaPhilippe Pasero
Apr 6, 2013·Journal of Molecular Biology·Hervé TécherBenoît Le Tallec
Jun 15, 2016·Proceedings of the National Academy of Sciences of the United States of America·Deepak BastiaMichael E O'Donnell
Sep 15, 2016·Small·Joris LacroixAurélien Bancaud
Dec 16, 2016·Scientific Reports·Emanuela TuminiAndrés Aguilera
Oct 24, 2017·Nucleic Acids Research·Elizabeth L ThompsonAlexandra Sobeck
Mar 29, 2019·Nucleic Acids Research·Nathaniel O WandRobert K Neely
Oct 28, 2017·The EMBO Journal·Irene Salas-ArmenterosAndrés Aguilera
Dec 3, 2014·The EMBO Journal·Irene Felipe-AbrioAndrés Aguilera
Mar 28, 2018·The EMBO Journal·Néstor García-RodríguezHelle D Ulrich
Feb 18, 2020·Cell Cycle·Belén Gómez-GonzálezAndrés Aguilera
Jun 5, 2019·Nature Communications·Philippe CoulombeMarcel Méchali
Feb 22, 2018·PLoS Genetics·Anthony PerrotPei-Yun Jenny Wu
Nov 19, 2013·Nature Structural & Molecular Biology·Silvana MourónJuan Méndez
Dec 13, 2016·Protoplasma·Marius Reinhart, M Cristina Cardoso
Nov 21, 2018·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Anna Ligasová, Karel Koberna
Sep 1, 2017·Nucleic Acids Research·Qing ZhangMarco Cosentino Lagomarsino
Aug 10, 2020·Nature Communications·Alexy PromonetPhilippe Pasero

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