Abstract
Total number and high-resolution position of DNA replication origins in Trypanosoma brucei remains unidentified until present days. Small nascent strand (SNS) quantification is the high-resolution technique for mapping of DNA replication origins. This method is based on the purification of single strand DNAs, primed with 10–12 ribonucleotides at 5’ end and synthesized by the DNA polymerase-α primase at origins of DNA replication. We have established isolation, purification and high-throughput sequencing of SNS (SNS-seq) in order to map the initiation sites of DNA replication (DNA replication origins) with high precision in procyclic and bloodstream-form of Trypanosoma brucei Lister 427 strain. We performed SNS-seq in these two life forms with three biological replicates each and we identified in total around 3800 replicated origin regions which corresponds to 110 sites / Mb genome. This number is in the same range as previously published for higher eukaryotes and other unicellular eukaryotes (Akermann 2020; Lombrana 2016; Besnard 2012). The origins of replication are organized as initiation sites or as initiation zones in both life forms and located predominantly in inter-genic regions. We found that 50% of the origin sites detected by SNS-seq are common between the two life forms. We are now analysing in detail the characteristics of the detected origin sites as well as their similarities and differences in these two life forms. In addition, we are establishing an episome-based assay to test if different types of DNA sequences can function as active DNA replication origins in Trypanosoma brucei Lister 427 strain. We want to use this system to functionally validate origin sites that were detected by SNS-seq, but also other types of sequencies like AT-rich, GC-rich or G4 structures. We have also analysed DNA replication parameters in these two life forms by DNA molecular combing that is following the progression of DNA synthesis at a single molecule resolution. We have found that the speed of replication forks and inter-origin distances are considerably different between procyclic and bloodstream-forms. The mean speed of replication forks is around 1.3x faster, while the mean inter-origin distance is 1.5x longer in bloodstream-forms compare to procyclic forms.