Stranded short nascent strand sequencing reveals the topology of DNA replication origins in Trypanosoma brucei

The universal features that define genomic regions acting as replication origins remain unclear. In this study, we mapped a set of origins in Trypanosoma brucei using stranded short nascent strand sequencing methods. Our results showed that DNA replication predominantly initiates in intergenic regions between poly(dA)- and poly(dT)-enriched sequences. G4 structures were detected in the vicinity of some origins and were embedded in poly(dA)-enriched sequences in a strand-specific manner: G4s on the plus strand were located upstream while those on the minus strand were located downstream of the centre. The origins' centres were found to be areas of low nucleosome occupancy, surrounded by regions of high nucleosome occupancy. Furthermore, our results demonstrate that 90% of replication origins overlap with a minor proportion of the previously reported RNA: DNA hybrids. These findings shed new light on the sequence and structural features that define the topology of replication origins in T. brucei . To further characterise replication dynamics at the single-molecule level, we employed DNA combing analysis.