Initiation of nuclear DNA replication in Trypanosoma brucei and Leishmania

Replication of the genome is a central process in cellular life, which must be tightly regulated at the risk of genomic instability. DNA replication has been extensively studied in bacteria and eukaryotes, and recently, in archaea. Universally, DNA replication is started at specific genomic sites termed origins of replication, which are recognised by an initiator factor. While in both bacteria and archaea the initiator factor is a single protein, it is assumed that this role is performed in eukaryotes by a highly conserved six-subunit origin recognition complex (ORC). Recent phylogenetic studies, however, suggest that the presence of a six-subunit ORC might not be as conserved as initially believed. Trypanosoma brucei is a protozoan parasite in which little is known about nuclear DNA replication. To date, initiation of T. brucei DNA replication has been associated with a single factor, TbORC1/CDC6, though highly diverged interacting partners have been identified. To elucidate whether T. brucei possesses a diverged ORC-like complex, TbORC1/CDC6 and its known interacting partners, TbORC1B, TbORC4, Tb7980, Tb3120, and a novel factor, Tb1120, were analysed. First, the protein sequences of these factors were re-analysed, revealing varying degrees of conservation and divergence with other eukaryotes’ ORC proteins. Second, expression silencing by inducible-RNA interference (RNAi) of TbORC1/CDC6, TbORC1B, TbORC4, and Tb3120, in procyclic form (PCF) and bloodstream form (BSF) cells, confirmed their involvement in DNA replication. Third, subcellular localisation and dynamics of TbORC1/CDC6 and its interacting partners during the cell cycle of PCF and BSF cells was investigated by immunofluorescence, revealing TbORC1B to be the sole factor to display an apparent cell cycle-dependent localisation pattern, perhaps suggesting that it might be a DNA replication regulatory factor. Finally, immunoprecipitation and gel filtration assays support the existence of an ORC-like complex, apparently large enough to be composed of TbORC1/CDC6 and known interacting partners, and potentially, additional factors. TbORC1/CDC6-binding sites and origins of replication in T. brucei have been mapped in PCF cells. Like most eukaryotes, no specific sequence elements were found to define TbORC1/CDC6-binding sites or origins of replication, and the TbORC1/CDC6-binding sites outnumbered the mapped origins, which appeared to be activated at different times during S phase. It has been reported in other eukaryotes that different cell types activate different origins or the activation timing differs. Here, origins of replication were mapped in T. brucei PCF and BSF cells, revealing a pronounced inflexibility in origin usage in these two life cycle stages. Only one, notable genome-wide difference was found: in BSF cells, the single active variant surface glycoprotein (VSG) expression site was found to be early replicating, whereas all other silent VSG expression sites were late replicating; in PCF cells however, where all VSG expression sites are silenced, these were all late replicating. These data reveal a locus-specific link between DNA replication and transcription in T. brucei, which may relate to immune evasion. The genomes of T. brucei and related kinetoplastids are highly syntenic. Since most eukaryotic origins of replication are not defined by consensus DNA sequences, but appear instead to be defined by, among other features, chromatin context and status, origins were here mapped in both L. major and L. mexicana promastigotes in order to ask if common features could be found relative to T. brucei. Surprisingly, only a single origin could be found per Leishmania chromosome, in contrast with all eukaryotes examined to date, where each chromosome is replicated from multiple detectable origins. Origin-active loci in Leishmania were found to be distinguishable from related non-origin loci in terms of size, a characteristic not observed in T. brucei, although around 40% of the mapped origins are conserved in location relative to T. brucei. These data reveal pronounced differences in replication dynamics between the two genera, despite the considerable overlap in genome organisation.