Identification of proteins involved in Trypanosoma brucei DNA replication fork dynamics using nascent DNA proteomics

DNA replication, transcription and chromatin remodeling are coordinated to ensure accurate duplication of genetic and epigenetic information. In regard to DNA replication, trypanosomatid parasites such as Trypanosoma brucei display unusual properties including significantly fewer origins of replication than model eukaryotes, a highly divergent Origin Replication Complex (ORC), and an apparent lack of several replication factor homologs. Although recent studies in T. brucei indicate functional links among DNA replication, transcription, and antigenic variation, the underlying mechanisms remain unknown. Here, we adapted an unbiased technology for the identification of replication fork proteins called iPOND (isolation of proteins on nascent DNA) to T. brucei, its first application to a parasite system. This led to the mass spectrometric identification of core replication machinery and of proteins associated with transcription, chromatin organization, and DNA repair that were enriched in the vicinity of an unperturbed active replication fork. Of a total of 410 enriched proteins, among which DNA polymerase α and replication factor C were scoring in the top, around 25% of the proteins identified were of unknown function and, therefore, have the potential to be essential trypanosome-specific replication proteins. Initial characterization of a protein annotated as a Replication Factor C subunit (Tb927.10.7990), and a protein of unknown function (Tb927.3.5370) revealed that both proteins retain nuclear localization throughout the cell cycle. While Tb927.3.5370 appeared to be a dispensable gene, Tb927.10.7990 proved to be essential since its silencing caused a growth defect in procyclic cells, accumulation of zoids and impaired DNA replication. Future studies on the generated proteins list can contribute to the understanding of DNA replication dynamics in T. brucei and how replication is coordinated with other cellular processes to maintain genome integrity.