Genome-wide identification and characterization of replication origins by deep sequencing

Carsten Russ,Hironori Niki,Chad Nusbaum,Keita Aoki,Naveen Kommajosyula,Jia Xu,Oliver J Rando,Nicholas Rhind,Patrice Milos,Alexander M Tsankov,Aviv Regev,Christopher Hart,James Bochicchio,Kathleen E Steinmann,Yoshimi Yanagisawa,Zhiping Weng

doi:10.1186/gb-2012-13-4-r27

Abstract

BackgroundDNA replication initiates at distinct origins in eukaryotic genomes, but the genomic features that define these sites are not well understood.ResultsWe have taken a combined experimental and bioinformatic approach to identify and characterize origins of replication in three distantly related fission yeasts: Schizosaccharomyces pombe, Schizosaccharomyces octosporus and Schizosaccharomyces japonicus. Using single-molecule deep sequencing to construct amplification-free high-resolution replication profiles, we located origins and identified sequence motifs that predict origin function. We then mapped nucleosome occupancy by deep sequencing of mononucleosomal DNA from the corresponding species, finding that origins tend to occupy nucleosome-depleted regions.ConclusionsThe sequences that specify origins are evolutionarily plastic, with low complexity nucleosome-excluding sequences functioning in S. pombe and S. octosporus, and binding sites for trans-acting nucleosome-excluding proteins functioning in S. japonicus. Furthermore, chromosome-scale variation in replication timing is conserved independently of origin location and via a mechanism distinct from known heterochromatic effects on origin function. These results are consistent with a model in which origins are simply the nucleosome-depleted regions of the genome with the highest affinity for the origin recognition complex. This approach provides a general strategy for understanding the mechanisms that define DNA replication origins in eukaryotes.

Highlights

DNA replication initiates at distinct origins in eukaryotic genomes, but the genomic features that define these sites are not well understood
Identification of origins by deep sequencing To identify origins in S. pombe, S. octosporus, and S. japonicus, we mapped all sites with increased DNA copy number in early S phase [11,24]
Our results are consistent with a general model for eukaryotic origin function in which origins are the nucleosomefree regions in the genome with the highest affinity for origin recognition complex (ORC) [4]

Summary

Introduction

DNA replication initiates at distinct origins in eukaryotic genomes, but the genomic features that define these sites are not well understood. Cis-acting sequences determine the location of replication origins in eukaryotic genomes [1]. The nature of such cis-acting sequences is not well understood. In the budding yeast Saccharomyces cerevisiae, the eukaryote with the best-studied origins, two sequence characteristics are important for origin function. In the fission yeast Schizosaccharomyces pombe, origins have been mapped genome-wide by a number of approaches [11,12,13,14,15]. These maps confirm earlier conclusions that origin function in S. pombe is conferred by high AT content [16,17]. NFRs at S. pombe origins have been both predicted and observed, due to the low resolution of existing origin maps and consequent heterogeneity in origin alignments, the averaged NFRs are broader and shallower than those in S. cerevisiae [4,21,22]

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Results

Discussion

Conclusion