Résumé
DNA replication is one of the most fundamental processes by which a cell makes a complete copy of its genome prior to cell division. DNA replication is initiated from multiple and distinct sites in the eukaryotic genome, but the universal features that define eukaryotic origins remain a mystery. Here, we applied stranded SNS-seq, a high-throughput sequencing method that preserves the directionality of short nascent strands (SNS), to map DNA replication origins in two life cycle stages of Trypanosoma brucei with unprecedented resolution. We performed an integrative study of features associated with origins and found that DNA replication starts predominantly in the intergenic regions, between poly(dA) and poly(dT) enriched sequences with point symmetry. Experimentally discovered G4 structures flanked 49-80% of origins in a specific manner: G4s on the plus strand are located upstream, whereas G4s on the minus strand are located downstream of the origin center. We found that G4 quadruplexes colocalize exclusively with poly(dA)-rich sequences near the origin center. The origin center is a nucleosome-depleted region flanked by nucleosome-enriched domains. We found that more than 90% of origins colocalized with previously reported R-loops, clearly linking R-loop formation and origins. We performed a DNA combing analysis, comparing DNA replication at the single cell level to the cell population level. We have, for the first time, determined the precise position of several building blocks of a eukaryotic origin and provided a comprehensive model.