Genome-scale analysis of metazoan replication origins reveals their organization in specific but flexible sites defined by conserved features

Abstract

In metazoans, thousands of DNA replication origins (Oris) are activated to replicate DNA at each cell cycle. Although their timing of activation is better understood, their genomic organization and their genetic nature remain elusive. Here, we identified Oris by nascent strand (NS) purification and characterized their common features by performing a genome-wide analysis in both Drosophila and mouse cell lines. We show that in both species most CpG islands (CGI) contain Oris, although methylation is nearly absent in Drosophila, indicating that this epigenetic mark is not crucial for defining the initiation event. Initiation of DNA synthesis starts at the borders of CGIs, resulting in a striking bimodal distribution of NS, suggestive of a dual initiation event. We also found that Oris contain a unique nucleotide skew around NS peaks, characterized by G/T and C/A over-representation at the 5' and 3' of Ori sites, respectively. Repeated GC-rich elements were detected, which are good predictors of Oris, suggesting that common sequence features are part of metazoan Oris. In the heterochromatic chromosome 4 of Drosophila, Oris are strongly correlated with HP1 binding sites. At the chromosome level, regions rich in Oris are early replicating, whereas Ori-poor regions co-localize with late replicating domains during the cell cycle. The genome-wide analysis was coupled with a DNA combing analysis to unravel the organization of replication origins. The results indicate that Oris are present in a large excess, but their activation does not occur at random. They are organized in groups of site-specific but flexible origins that define replicons, where a single origin is activated in each replicon. This organization provides both site specificity and Ori firing flexibility in each replicon, allowing possible adaptation of DNA replication to environmental cues and cell fates.