Authors

Discussion

      Nuclear DNA replication is initiated at specific genomic loci termed origins. In eukaryotes, these sites are defined by the binding of the six-subunit Origin Recognition Complex (ORC). Frequently described as a well-conserved complex, phylogenetic studies querying a wide variety of species from across the eukaryotic tree suggest that many may have lost one or more ORC subunits. In Trypanosoma brucei, four ORC subunits (ORC1/CDC6, ORC4, ORC5 and ORC2) have been identified to date, most of which diverge considerably from their eukaryotic orthologues. The two remaining canonical subunits, Orc3 and Orc6, remain unidentified, and it is unclear whether a six-subunit ORC exists in T. brucei. 

      To elucidate the composition of TbORC, we have resorted to two main approaches: bioinformatics and experimental. Using publicly available genome and transcriptome datasets, we have run extensive sequence-based homology searches. Orc3 orthologues were detected in most analysed Discoba lineages but not in kinetoplastids, suggesting that this subunit might be missing in this group. Immunoprecipitation of multiple TbORC subunits resulted in the common identification of Tb1120 (Tb927.6.1120), and gel filtration analysis showed that Tb1120 elutes in fractions of similar molecular weight to those of other TbORC subunits. Moreover, RNAi depletion resulted in severe growth arrest, cell cycle de-regulation, and almost complete abrogation of DNA replication, akin to the effects of depletion of other TbORC subunits. Protein domain searches and de novo structure predictions suggest that the only similarity between Tb1120 and canonical ORC subunits is a putative C-terminal winged helix-turn-helix domain (commonly associated with DNA binding and/or protein-protein interactions). Sequence-based homology searches failed to detect evidence for a relationship between Tb1120 and any known ORC subunit, and were unable to find any orthologues of Tb1120 outside of the kinetoplastids, raising the hypothesis of it replacing Orc3 in TbORC. If correct, incorporation of a kinetoplastid-specific ORC subunit may have necessitated wider reorganisation of the kinetoplastid ORC, consistent with the considerable divergence in ORC2 and ORC5 as described previously. The biological reasons behind kinetoplastids incorporating a putative novel subunit into their ORC are unknown, but are the subject of ongoing studies.