A distinct mode of DNA replication initiation in trypanosomes?

The growth and propagation of all living organisms requires the faithful transmission of their genome, as this genetic material provides the blueprint for life. Transmission of the genome from parent to offspring requires that the genetic material be copied and then segregated. In all cellular organisms the genome is composed of DNA, and its copying is referred to as DNA replication. In eukaryotic cells (e.g. humans and yeast), replication of the DNA genome is a highly regulated, multi-step process. DNA replication starts with the binding of a specific protein complex, the 'initiator', to specific regions of the genome. These regions are called origins and are the sites where copying of the DNA begins. Binding of the initiator to the origins leads to a series of events that result in the recruitment of other players to the origins, including another protein complex called the 'helicase'; this complex opens the DNA at the origin and allows the copying machinery (e.g. DNA polymerases) to travel along the DNA, copying each strand to generate two near-identical copies of the original DNA. The helicase is not, however, recruited to the origin directly by the initiator, but through two mediator proteins. In most eukaryotes organisms that have been studied, the initiator, helicase and the two mediators are present and are quite similar. Furthermore, all these proteins are recruited to the origins at a specific stage of the cell's growth, prior to the stage where DNA replication takes place. In African trypanosomes, however, both these aspects of DNA replication seem to be different.

African trypanosomes are single cell eukaryote microbes that cause severe and debilitating disease in both humans and other animals (such as cattle) in sub-Saharan Africa. The results of our work (and from others) suggest three things: that the trypanosome's initiator looks very different from the protein complex seen in other eukaryote organisms (human, fly and yeast); it is unclear if the two mediators exist in these microbes; and that the timing of interaction between the initiator, the mediator proteins (if they exist) and the helicase occurs at a distinct stage of cell growth from that seen in other eukaryotes.

With this project, we want to explore the differences mentioned above by following a number of lines of investigation. First, we have found a protein that interacts with the initiator, but it is not similar to any of the initiator components in other eukaryotes; we want to find out if it is part of the initiator and how it affects DNA replication. Second, as it is unclear if trypanosomes have the two mediators, we want to test if two proteins identified in trypanosomes provide these functions and how do they do so. Finally, we want to explore the dynamics of the timing of interaction between the initiator, the putative mediator proteins, and the helicase which, as mentioned above, seems to take place at a different stage of cell growth from other eukaryotes. To perform these analyses, we will use a wide range of cellular and genetic approaches.

If our hypotheses are correct, our findings will alter the prevailing view of DNA replication regulation across the diverse range of eukaryotic organisms, and open new lines of research. In addition, our work will reveal how DNA replication connects with a wide range of other cellular processes in trypanosomes, including how they survive in the host and express the content of their genome. Importantly, and in the long term, if the players in DNA replication in trypanosomes are indeed very different from those in their hosts (humans and animals), our findings will provide a group of potential targets for the development new treatments for parasitic diseases.

DNA replication is a central cellular reaction, necessary for genome transmission and the propagation of life. Structural and mechanistic studies on DNA replication have focused mainly on a small number of eukaryotes, revealing conserved machinery and reactions. However, data from Trypanosoma brucei, a diverged eukaryotic microbe and major parasite, suggest the machinery, timing and regulation of DNA replication initiation is 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 eukaryotes so far studied, and instead occurs during S phase. We will test this hypothesis through four objectives:

1. 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.

2. 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.

3. 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.

4. 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.