Though no work to date has examined the link between nuclear DNA replication and cell cycle checkpoints in T. brucei, perturbation of ORC function activates DNA damage and spindle checkpoints in yeast. Despite this, we cannot yet exclude the possibility that the RNAi phenotypes we see reflect non-replication functions for TbORC1/CDC6 and for the ORC1/CDC6-interacting factors, and perhaps the greater severity of RNAi in BSF cells is because these non-replication roles assume greater prominence in mammal life cycle stages. An intriguing feature of ORC in T. brucei, as well as in G. lamblia and N. gruberi, is the putative absence of distinct Orc1 and Cdc6 factors. Orc1 and Cdc6 are very similar in sequence and so it is highly unlikely that database searches would have found one gene but not the other, unless one had diverged significantly in sequence. As it has been argued, the fusion of Orc1 and Cdc6 functions in a single protein may be representative of an ancestral molecule, as is found in archaea. However, Dabrafenib 1195765-45-7it must now be considered that T. brucei ORC1/CDC6 does not function in isolation, but as part of an ORC containing at least one other factor. The functional consequences of this unusual arrangement are unclear. In other eukaryotes, Cdc6 becomes associated with ORC in a cell cycle-specific manner, inducing ATP-dependent conformational changes that cause recruitment of the MCM helicase, via Cdt1. Expression of T. brucei and T. cruzi ORC1/CDC6 is not cell cycle-dependent; instead the protein associates with chromatin in all cell cycle stages. This appears to rule out the possibility that TbORC1/CDC6 is functionally related to Cdc6 and not Orc1. However, if Cdc6 is constitutively fused to ORC, this raises questions regarding how and when the MCM replicative helicase is recruited, with implications for the regulation of origin firing in these parasites. This is a problem shared with archaea, and we considered the possibility that T. brucei ORC might interact directly with MCM. While our interaction data are compatible with T. brucei MCM possessing a conventional eukaryotic heterohexameric MCM structure with a conserved subunit interaction network, we were unable to detect interactions between TbORC1/CDC6 and TbMCM,GSI-IXusing a number of approaches. More specifically, IP of HA-tagged TbMCM3, TbMCM6 or TbMCM7 did not reveal coIP of myc-tagged TbORC1/CDC6 from whole cells extracts. Conversely, IP of myc-tagged TbORC1/CDC6 did not coIP HAtagged TbMCM2 or TbMCM7. Finally, IP and mass-spectrometry analyses of HA-tagged MCM subunits or Myc-tagged TbORC1/CDC6 did not reveal evidence for interaction.