The dynamics of genome replication using deep sequencing

TY - JOUR

T1 - The dynamics of genome replication using deep sequencing

AU - Müller, Carolin A

AU - Hawkins, Michelle

AU - Retkute, Renata

AU - Malla, Sunir

AU - Wilson, Ray

AU - Blythe, Martin J

AU - Nakato, Ryuichiro

AU - Komata, Makiko

AU - Shirahige, Katsuhiko

AU - de Moura, Alessandro P S

AU - Nieduszynski, Conrad A

PY - 2014/1/1

Y1 - 2014/1/1

N2 - 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.

AB - 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.

KW - DNA Replication

KW - Genome

KW - High-Throughput Nucleotide Sequencing

KW - Replication Origin

KW - Saccharomyces cerevisiae

KW - Sequence Analysis, DNA

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1093/nar/gkt878

DO - 10.1093/nar/gkt878

M3 - Article

C2 - 24089142

SN - 0305-1048

VL - 42

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 1

M1 - e3

ER -