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

Julien N. Bianco; Jérôme Poli; Julie Saksouk; Julien Bacal; Maria Joao Silva; Kazumasa Yoshida; Yea Lih Lin; Hélène Tourrière; Armelle Lengronne; Philippe Pasero

doi:10.1016/j.ymeth.2012.04.007

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

Julien N. Bianco, Jérôme Poli, Julie Saksouk, Julien Bacal, Maria Joao Silva, Kazumasa Yoshida, Yea Lih Lin, Hélène Tourrière, Armelle Lengronne, Philippe Pasero

Research output: Contribution to journal › Article › peer-review

78 Citations (Scopus)

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.

Original language	English
Pages (from-to)	149-157
Number of pages	9
Journal	Methods
Volume	57
Issue number	2
DOIs	https://doi.org/10.1016/j.ymeth.2012.04.007
Publication status	Published - Jun 2012
Externally published	Yes

All Science Journal Classification (ASJC) codes

Molecular Biology
Biochemistry, Genetics and Molecular Biology(all)

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10.1016/j.ymeth.2012.04.007

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@article{0a976123dad7411eb691b5da37ac6272,

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.",

author = "Bianco, {Julien N.} and J{\'e}r{\^o}me Poli and Julie Saksouk and Julien Bacal and Silva, {Maria Joao} and Kazumasa Yoshida and Lin, {Yea Lih} and H{\'e}l{\`e}ne Tourri{\`e}re and Armelle Lengronne and Philippe Pasero",

note = "Funding Information: We thank Grant Brown (U. Toronto) for the pRS415 plasmid expressing pADH-hENT1 and Oscar Aparicio (U. Southern California) for the HSV-TK-hENT1 integrative vector. Work in the Pasero lab is supported by ANR and by Fondation pour la Recherche M{\'e}dicale (Equipe FRM) and by fellowships from ARC to JB and from the French “Minist{\`e}re de la Recherche et de l{\textquoteright}Enseignement Sup{\'e}rieur” to JP. MJS thanks CNRS and the Region Languedoc-Roussillon for support. We also thank E. Schwob and the DNA combing facility of Montpellier for providing silanized coverslips and the Montpellier RIO Imaging microscopy facility.",

year = "2012",

month = jun,

doi = "10.1016/j.ymeth.2012.04.007",

language = "English",

volume = "57",

pages = "149--157",

journal = "ImmunoMethods",

issn = "1046-2023",

publisher = "Academic Press Inc.",

number = "2",

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TY - JOUR

T1 - Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing

AU - Bianco, Julien N.

AU - Poli, Jérôme

AU - Saksouk, Julie

AU - Bacal, Julien

AU - Silva, Maria Joao

AU - Yoshida, Kazumasa

AU - Lin, Yea Lih

AU - Tourrière, Hélène

AU - Lengronne, Armelle

AU - Pasero, Philippe

N1 - Funding Information: We thank Grant Brown (U. Toronto) for the pRS415 plasmid expressing pADH-hENT1 and Oscar Aparicio (U. Southern California) for the HSV-TK-hENT1 integrative vector. Work in the Pasero lab is supported by ANR and by Fondation pour la Recherche Médicale (Equipe FRM) and by fellowships from ARC to JB and from the French “Ministère de la Recherche et de l’Enseignement Supérieur” to JP. MJS thanks CNRS and the Region Languedoc-Roussillon for support. We also thank E. Schwob and the DNA combing facility of Montpellier for providing silanized coverslips and the Montpellier RIO Imaging microscopy facility.

PY - 2012/6

Y1 - 2012/6

N2 - 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.

AB - 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.

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EP - 157

JO - ImmunoMethods

JF - ImmunoMethods

IS - 2

ER -

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

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