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The dynamics of genome replication using deep sequencing

M�ller, 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.

Authors

Carolin A. M�ller

Michelle Hawkins

Renata Retkute

Sunir Malla

Ray Wilson

Martin J. Blythe

Ryuichiro Nakato

Makiko Komata

Katsuhiko Shirahige

Alessandro P.S. de Moura

Conrad A. Nieduszynski



Abstract

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.

Citation

Müller, C. A., Hawkins, M., Retkute, R., Malla, S., Wilson, R., Blythe, M. J., …Nieduszynski, C. A. (2014). The dynamics of genome replication using deep sequencing. Nucleic Acids Research, 42(1), Article e3. https://doi.org/10.1093/nar/gkt878

Journal Article Type Article
Publication Date Jan 1, 2014
Deposit Date Apr 15, 2014
Publicly Available Date Apr 15, 2014
Journal Nucleic Acids Research
Print ISSN 0305-1048
Electronic ISSN 0305-1048
Publisher Oxford University Press
Peer Reviewed Peer Reviewed
Volume 42
Issue 1
Article Number e3
DOI https://doi.org/10.1093/nar/gkt878
Public URL https://nottingham-repository.worktribe.com/output/997556
Publisher URL http://nar.oxfordjournals.org/content/42/1/e3.long

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