The dynamics of genome replication using deep sequencing

Carolin A Müller, 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

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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.
Original languageEnglish
Article numbere3
JournalNucleic Acids Research
Volume42
Issue number1
Early online date1 Oct 2013
DOIs
Publication statusPublished - Jan 2014

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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), [e3]. https://doi.org/10.1093/nar/gkt878