Structural analysis of the origin recognition complex (ORC) in Trypanosoma brucei using cryo-electron microscopy and single-particle analysis

Abstract

DNA replication is a biological process of paramount importance, mainly because it drives the propagation of all living organisms, being the basis of genetic inheritance. The earliest step of DNA replication is the establishment of origins, which are defined as genomic loci where DNA synthesis initiates, being preceded by the binding of a replication initiation factor. The binding of the initiator establishes replication initiation by the recruitment and activation of the replisome. In prokaryotes, a single initiator binds each origin, whereas in eukaryotes a six-protein origin recognition complex, termed Origin Recognition Complex (ORC), play this function in conjunction with a related factor called Cdc6. The evolutionary pathways that led eukaryotes to evolve a multisubunit initiator are unknown, as well as the roles played by each ORC subunit. One way to address this is to functionally characterize ORC from a diverged eukaryote since most studies to date have been limited in phylogenetic breadth. Trypanosoma brucei, a unicellular eukaryote parasite with peculiar genetic tools, provides a means to tackle this question. Sequence-based searches failed to identify ORC components in T. brucei, but biochemical approaches have now described four putative ORC subunits (ORC1/CDC6, ORC4, Tb3120, and Tb7980). At least two are present in a high molecular complex and all act in nuclear DNA replication, but each one displays considerable sequence divergence from 'model' ORC subunits. Moreover, another ORC-like replication factor (ORC1B) has been identified as a non-static constituent of ORC, displaying highly unusual S-phase restricted nuclear localization or expression, suggesting a possible new model to regulate the replication. These findings raise questions about the constitution and mode of action of ORC in T. brucei that may shed light on ORC evolution, which may provide a route for future drug development. Thus, this project aims to determine the complete architecture of ORC in T. brucei, comparing it to previously published structures from humans and Drosophila. To achieve this aim, we will perform immunoprecipitation (IP) of native ORC from T. brucei followed by cryo-electron microscopy (cryo-EM) and single-particle analysis (SPEM). Additionally, we will co-express and purify the known T. brucei ORC components, and use cryo-EM to ask how these interact, comparing them with known ORCs. Of note, cryo-EM and SPEM applied to determine structures with high resolution (< 4Å) from IP is a labor-intensive technique still not very widespread in Brazil, which makes this project an excellent opportunity to bring this approach to my host country and apply it to other trypanosomatids that cause endemic diseases, such as Chagas disease. (AU)