This finding suggests that an ORC is present in T. brucei, though it is highly diverged. In support of this, we show that RNAi knockdown TbORC1/CDC6 or any of the three interacting factors results in striking phenocopying in both tsetse fly- and mammal-infective T. brucei, suggesting functional overlap. Finally, we describe a sub-complex of the T. brucei MCM helicase, which suggests a conserved topology with other eukaryotic MCM helicases. In eukaryotes Cdc6 and Cdt1 function to recruit the MCM helicase complex for local DNA unwinding by mediating interaction with ORC. Homology searches have failed to identify a Cdt1 homologue in any trypanosomatid genome. A mechanistic consequence of such a putative absence could be that MCM in T. brucei is recruited directly by BEZ235 915019-65-7 TbORC1/ CDC6. If correct, this would lend support to the suggestion that the very earliest steps in origin designation in T. brucei may be archaeal-like, and may relate to the potential that TbORC1/ CDC6 provides both Cdc6 and Orc1 functions. Such direct interaction between one or more subunits of ORC and MCM has not been reported in eukaryotes, but has been seen between archaeal ORC1/CDC6 and the replicative helicase in a number of species by different methods. We therefore decided to test this experimentally in T. brucei. Unlike in archaea, where the replicative helicase is a homohexamer, orthologues of all six core eukaryotic MCM subunits, named Mcm2–7, can be unambiguously identified in the T. brucei genome. In addition, putative MCM8 and MCM9BIBW2992 orthologues appear to be present. To test if TbORC1/CDC6 directly interacts with the MCM2–7 helicase, we generated constructs that allow us to C-terminally tag TbORC1/CDC6 with 12 copies of the Myc epitope, and to Cterminally tag each of the MCM subunits with six copies of the HA epitope. We first generated procyclic form cells expressing TbORC1/CDC6-Myc, and these were then transformed individually with the six MCM subunit tagging constructs. Clones were obtained that expressed proteins reacting with anti- HA antiserum that were of the size expected for HA-tagged variants of each of the TbMCM subunits. In each case, co-expression of TbORC1/CDC6-Myc was confirmed by hybridisation with anti-Myc antibody. From the western blots it is evident that the individual HA-tagged TbMCM subunits were detected at different levels by the anti-HA antisera. Visual inspection indicated TbMCM2 generated the weakest signal, followed by TbMCM7, and TbMCM3, -4 and -6 gave the same, higher levels. Clones expressing HA-MCM5 were recovered later and the level of expression of this protein appeared low relative to the other MCM subunits ; these clones were not analysed further.