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

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.

Controlling DNA replication origins in response to DNA damage – inhibit globally, activate locally

“…caused by underlying differences in the efficiencies of origins (Bechhoefer and Rhind, 2012;Rhind et al, 2010;Xu et al, 2012). Origin timing is a two-step process: the temporal programme is first determined early in the cell cycle (Dimitrova and Gilbert, 1999;Raghuraman et al, 1997), probably by establishing specific chromatin states around origins (see Box 2), and second, the time of origin firing during S phase is determined by the affinity of origins for limiting amounts of firing factors (Box 3).…”

Characterizing and controlling intrinsic biases of lambda exonuclease in nascent strand sequencing reveals phasing between nucleosomes and G-quadruplex motifs around a subset of human replication origins

“…What defines origins in metazoans remains unclear. Origins are AT-rich in bacteria and yeast (Méchali 2010;Leonard and Méchali 2013) with a few exceptions (Xu et al 2012;Liachko et al 2014). Similarly, many origins in metazoans have AT-rich elements (Aladjem and Fanning 2004).…”

Chemical map of Schizosaccharomyces pombe reveals species-specific features in nucleosome positioning

“…The fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae are two highly diverged model organisms widely used to study basic biological processes in eukaryotes (23). Although similar in size, S. pombe is distinguished from S. cerevisiae by sharing important characteristics of chromosome structure with metazoans, including relatively large chromosomes, numerous introns, large repetitive centromeres, low-complexity replication origins, the composition of chromatin remodeling complexes, and siRNA-regulated heterochromatin (24)(25)(26)(27). Recent micrococcal nuclease (MNase) mapping studies (16,28,29) revealed substantial differences between the two species in nucleosomal DNA sequence features and chromatin organization, including how histone-DNA sequence preferences affect nucleosome positioning and nucleosome occupancy patterns around transcription start sites (TSSs) and DNA replication origins.…”