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Open Access Dissertation
Doctor of Philosophy (PhD)
Year Degree Awarded
Month Degree Awarded
Michele M Klingbeil
Yasu S. Morita
M. Sloan Siegrist
Molecular Biology | Parasitology | Pathogenic Microbiology
DNA is the substrate of many cellular processes including DNA replication, transcription and chromatin remodeling. These processes are coordinated to maintain genome integrity and ensure accurate duplication of genetic and epigenetic information. Genome-wide studies have provided evidence of the relationship between transcription and DNA replication timing. A global analysis of DNA replication initiation in T. brucei showed that TbORC1 (subunit of the origin recognition complex, ORC) binding sites are located at the boundaries of transcription units. Although recent studies in T. brucei indicate functional links among DNA replication and transcription, the underlying mechanisms remain unknown. In this study, we adapted an unbiased technology for the identification of replication fork proteins called iPOND (isolation of proteins on nascent DNA) to T. brucei, its first application to a parasite system.The iPOND approach relies on labeling newly replicated DNA with the thymidine analog EdU (5-ethynyl-2′-deoxyuridine), which contains an alkyne functional group that enables the cycloaddition of a biotin azide. This click chemistry reaction yields a stable covalent linkage, facilitating streptavidin capture of cross-linked biotinylated DNA-protein complexes. First, we described how we adapted the iPOND protocol in T. brucei cells to generate a suitable sample for mass spectrometry analysis (Chapter 2). We performed MS label-free quantification to determine which proteins are enriched in an active replication fork in T. brucei (Chapter 3). We identified 410 proteins, including key DNA replication factors and proteins associated with transcription, chromatin organization, DNA repair and mRNA splicing. Around 25% of the proteins identified were of unknown function that might have the potential to be essential trypanosome-specific replication proteins. Additionally, we characterized two proteins from our iPOND-derived protein list (Chapter 3). These are a protein annotated as a Replication Factor C subunit (Tb927.10.7990), and a protein of unknown function (Tb927.3.5370). Both revealed nuclear localization. Tb927.10.7990 proved to be essential since its silencing caused a growth defect and impaired DNA replication. However,Tb927.3.5370 appeared to be a dispensable gene. We propose nucleosomes are assembled close to the replication fork followed by RNA pol II recruitment, transcription, and co-transcriptional RNA splicing. Further studies are needed to determine how these processes are linked and co-regulated, and how rapidly they are initiated during DNA replication.
Rocha Granados, Maria, "NASCENT DNA PROTEOMICS ANALYSIS UNCOVERS DNA REPLICATION DYNAMICS IN THE HUMAN PATHOGEN TRYPANOSOMA BRUCEI" (2019). Doctoral Dissertations. 1492.