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The dynamics of genome replication using deep sequencing

Eukaryotic genomes are replicated from multiple DNA replication origins. We present complementary deep sequencing approaches to measure origin location and activity in Saccharomyces cerevisiae. Measuring the increase in DNA copy number during a synchronous S-phase allowed the precise determination o...

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Autores principales: Müller, Carolin A., Hawkins, Michelle, Retkute, Renata, Malla, Sunir, Wilson, Ray, Blythe, Martin J., Nakato, Ryuichiro, Komata, Makiko, Shirahige, Katsuhiko, de Moura, Alessandro P.S., Nieduszynski, Conrad A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3874191/
https://www.ncbi.nlm.nih.gov/pubmed/24089142
http://dx.doi.org/10.1093/nar/gkt878
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author Müller, Carolin A.
Hawkins, Michelle
Retkute, Renata
Malla, Sunir
Wilson, Ray
Blythe, Martin J.
Nakato, Ryuichiro
Komata, Makiko
Shirahige, Katsuhiko
de Moura, Alessandro P.S.
Nieduszynski, Conrad A.
author_facet Müller, Carolin A.
Hawkins, Michelle
Retkute, Renata
Malla, Sunir
Wilson, Ray
Blythe, Martin J.
Nakato, Ryuichiro
Komata, Makiko
Shirahige, Katsuhiko
de Moura, Alessandro P.S.
Nieduszynski, Conrad A.
author_sort Müller, Carolin A.
collection PubMed
description Eukaryotic genomes are replicated from multiple DNA replication origins. We present complementary deep sequencing approaches to measure origin location and activity in Saccharomyces cerevisiae. Measuring the increase in DNA copy number during a synchronous S-phase allowed the precise determination of genome replication. To map origin locations, replication forks were stalled close to their initiation sites; therefore, copy number enrichment was limited to origins. Replication timing profiles were generated from asynchronous cultures using fluorescence-activated cell sorting. Applying this technique we show that the replication profiles of haploid and diploid cells are indistinguishable, indicating that both cell types use the same cohort of origins with the same activities. Finally, increasing sequencing depth allowed the direct measure of replication dynamics from an exponentially growing culture. This is the first time this approach, called marker frequency analysis, has been successfully applied to a eukaryote. These data provide a high-resolution resource and methodological framework for studying genome biology.
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spelling pubmed-38741912013-12-28 The dynamics of genome replication using deep sequencing Müller, Carolin A. Hawkins, Michelle Retkute, Renata Malla, Sunir Wilson, Ray Blythe, Martin J. Nakato, Ryuichiro Komata, Makiko Shirahige, Katsuhiko de Moura, Alessandro P.S. Nieduszynski, Conrad A. Nucleic Acids Res Methods Online Eukaryotic genomes are replicated from multiple DNA replication origins. We present complementary deep sequencing approaches to measure origin location and activity in Saccharomyces cerevisiae. Measuring the increase in DNA copy number during a synchronous S-phase allowed the precise determination of genome replication. To map origin locations, replication forks were stalled close to their initiation sites; therefore, copy number enrichment was limited to origins. Replication timing profiles were generated from asynchronous cultures using fluorescence-activated cell sorting. Applying this technique we show that the replication profiles of haploid and diploid cells are indistinguishable, indicating that both cell types use the same cohort of origins with the same activities. Finally, increasing sequencing depth allowed the direct measure of replication dynamics from an exponentially growing culture. This is the first time this approach, called marker frequency analysis, has been successfully applied to a eukaryote. These data provide a high-resolution resource and methodological framework for studying genome biology. Oxford University Press 2014-01-01 2013-10-01 /pmc/articles/PMC3874191/ /pubmed/24089142 http://dx.doi.org/10.1093/nar/gkt878 Text en © The Author(s) 2013. Published by Oxford University Press. http://creativecommons.org/licenses/by/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Methods Online
Müller, Carolin A.
Hawkins, Michelle
Retkute, Renata
Malla, Sunir
Wilson, Ray
Blythe, Martin J.
Nakato, Ryuichiro
Komata, Makiko
Shirahige, Katsuhiko
de Moura, Alessandro P.S.
Nieduszynski, Conrad A.
The dynamics of genome replication using deep sequencing
title The dynamics of genome replication using deep sequencing
title_full The dynamics of genome replication using deep sequencing
title_fullStr The dynamics of genome replication using deep sequencing
title_full_unstemmed The dynamics of genome replication using deep sequencing
title_short The dynamics of genome replication using deep sequencing
title_sort dynamics of genome replication using deep sequencing
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3874191/
https://www.ncbi.nlm.nih.gov/pubmed/24089142
http://dx.doi.org/10.1093/nar/gkt878
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