The dynamics of genome replication using deep sequencing.

Carolin A Müller,Renata Retkute,Makiko Komata,Sunir Malla,Ryuichiro Nakato,Michelle Hawkins,Martin J Blythe,Ray Wilson,Conrad A Nieduszynski,Alessandro P.S De Moura,Katsuhiko Shirahige

doi:10.1093/nar/gkt878

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.

Highlights

Complete replication of the genome is a requirement for successful cell division and is fundamental to all life
Checkpoint-deficient S. cerevisiae cells were synchronized in G1-phase and released into S-phase in the presence of the ribonucleotide reductase inhibitor HU
Replicating (S-phase) and nonreplicating (G2 phase) cells were selected by fluorescence-activated cell sorting (FACS)

Summary

Introduction

Complete replication of the genome is a requirement for successful cell division and is fundamental to all life. The efficient replication of eukaryotic genomes requires coordinated initiation from thousands of DNA replication origins [1,2]. Such coordinated origin activation leads to stereotypic replication-timing programs in which replication timing correlates with chromatin structure, gene expression, genome evolution and other critical aspects of genome metabolism. Several mutations have been characterized that disrupt the temporal order of genome replication. Some of these mutants globally alter replication timing, either by changes to chromatin modifications or subnuclear positioning [5,6]. Mutants have been isolated that change the replication time of telomeres from late to early S-phase [7,8] and different mutants that delay the normally early replication time of centromeres [9]

Methods

Results

Conclusion