Genome-wide identification and characterization of replication origins by deep sequencing
1 Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA
2 Bioinformatics Core Facility, University of Massachusetts Medical School, Worcester, MA 01605, USA
3 Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA
4 Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA 02142, USA
5 Helicos BioSciences Corporation, Cambridge, MA 02139, USA
6 Microbial Genetics Laboratory, Genetic Strains Research Center, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan
7 Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, MD 20815-6789, USA
8 Division of Natural Sciences, New College of Florida, Sarasota, FL 34243, USA
9 Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA 02142, USA
Genome Biology 2012, 13:R27 doi:10.1186/gb-2012-13-4-r27Published: 24 April 2012
DNA replication initiates at distinct origins in eukaryotic genomes, but the genomic features that define these sites are not well understood.
We 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.
The 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.