DNA replication is a central cellular reaction, necessary for genome transmission and the propagation of life. To date, structural and mechanistic studies on DNA replication have focused mainly on a small number of eukaryotes, revealing conserved machinery and reactions. However, emerging data from Trypanosoma brucei, a diverged eukaryotic microbe and major parasite, suggest the machinery, timing and regulation of DNA replication initiation may be distinct from this generalized model. We hypothesise that assembly of the T. brucei DNA replication initiation machinery is deferred from the G1 phase of the cell cycle, as seen in all eukaryotes so far studied, and instead occurs during S phase. We will test this hypothesis through four objectives:
- DNA replication initiates in eukaryotes through the binding of a six subunit Origin Recognition Complex (ORC) to genomic loci termed origins. Previous data suggest that T. brucei ORC deviates from the conventional six-subunit architecture. We will test if this deviation is due to the action of a newly discovered, novel T. brucei ORC subunit that is only found in close relatives of T. brucei.
- Once bound to origins, ORC recruits the Minichromosome Maintenance (MCM) replicative helicase in G1, a reaction guided by two mediator factors, Cdc6 and Cdt1. To date, no discrete Cdc6-like protein has been described in T. brucei and so we will test if this activity resides in a protein termed TbORC1B, whose expression is limited to S phase.
- In other eukaryotes, MCM is assembled and recruited to ORC on origins in G1 of the cell cycle. We will test recent data suggesting that at least some subunits of MCM are only expressed in S phase, and if this means that MCM-ORC interaction is deferred until this cell cycle stage.
- No work has yet described a Cdt1-like mediator in T. brucei. We will test if this activity resides in a recently discovered, highly diverged Cdt1-related factor, including whether this activity is limited to S phase.