Study of how species-specific interactions with the proliferating cell nuclear antigen of Trypanosoma brucei impact DNA replication

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Proliferating cell nuclear antigen (PCNA) is conserved in all eukaryotes. The knowledge about PCNA interactions and how they regulate DNA replication in eukaryotes comes mostly from the studies in yeast and animal cells. This project will study PCNA interactions in a different lineage of eukaryotes, the single-cell parasite Trypanosoma brucei, representing protists, the most diverse group of eukaryotes. The PCNA of T. brucei has a unique added structure element, a large backside loop that is phosphorylated by a mitogen-activated protein kinase called TbERK8. Such an interaction does not occur in the PCNA homologs from yeast or animal cells. The importance of this study is in its impact on increasing fundamental knowledge of the evolution and diversity of mechanisms used by PCNA to regulate DNA replication in eukaryotes. This project will provide experiential training to undergraduate and graduate students from underrepresented groups and Historically Black Universities and Colleges (HBCUs) to help train the next generation of diverse basic researchers.Many studies show that PCNA functions as a master regulator of DNA replication by way of interactions with other proteins. Thus far, PCNA functions were mostly investigated using standard models such as yeast and animal cells, which belong to the same lineage of eukaryotes. Moreover, all the PCNA interactions studied to date occur on its front face. This project will uncover new knowledge about eukaryotic DNA replication using two strategies: Firstly, it will use T. brucei, a protist representing the most diverse group of eukaryotes; trypanosomatids split from the eukaryotic ancestor lineage before yeast or metazoa, and their DNA replication machinery and mechanisms of regulation are poorly understood. Secondly, this project will focus on the large backside loop of TbPCNA that gets phosphorylated on serine and threonine residues and associates with other backside loop-associated proteins (BSL-APs). This project has the potential to advance knowledge of eukaryotic DNA replication by studying how the phosphorylation status of the backside loop and its interaction with BSL-APs regulate DNA replication in T. brucei. Examining how two uncharacterized trypanosome-specific BSL-APs interact with the TbPCNA backside loop will advance knowledge of novel gene functions involved in eukaryotic DNA replication.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.