title: Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing.
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.
authors.affiliation: Institute of Human Genetics, CNRS UPR 1142, Montpellier F-34396, France.
authors.forename: Julien N
authors.initials: JN
authors.lastname: Bianco
authors.forename: Jérôme
authors.initials: J
authors.lastname: Poli
authors.forename: Julie
authors.initials: J
authors.lastname: Saksouk
authors.forename: Julien
authors.initials: J
authors.lastname: Bacal
authors.forename: Maria Joao
authors.initials: MJ
authors.lastname: Silva
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Yoshida
authors.forename: Yea-Lih
authors.initials: YL
authors.lastname: Lin
authors.forename: Hélène
authors.initials: H
authors.lastname: Tourrière
authors.forename: Armelle
authors.initials: A
authors.lastname: Lengronne
authors.forename: Philippe
authors.initials: P
authors.lastname: Pasero
mesh_terms: D000818:Animals; D001973:Bromodeoxyuridine / Q000378:metabolism; D057930:Click Chemistry; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D004261:DNA Replication; D004271:DNA, Fungal / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D004277:DNA, Single-Stranded / Q000737:chemistry; D003627:Data Interpretation, Statistical; D019084:Fluorescent Antibody Technique, Indirect; D016681:Genome, Fungal; D015894:Genome, Human; D045325:HCT116 Cells; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D061925:Immobilized Nucleic Acids / Q000737:chemistry; D017404:In Situ Hybridization, Fluorescence; D008322:Mammals; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013194:Staining and Labeling; D018709:Statistics, Nonparametric
chemical_list: D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D061925:Immobilized Nucleic Acids; D019384:Nucleic Acid Synthesis Inhibitors; D004247:DNA; D001973:Bromodeoxyuridine; D006918:Hydroxyurea