Investigating the role of Plasmodium falciparum apicoplast DNA polymerase in plastid DNA replication and repair

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Nieto, Nicholas Stephen
Major Professor
Nelson, Scott W
Beck, Joshua R
Honzatko, Richard B
Andreotti, Amy H
Shogren-Knaak, Michael A
Committee Member
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Biochem, Biophysics, and Molecular Biology
Malaria is a deadly and ancient disease caused by unicellular eukaryotic parasites of the genus plasmodium with falciparum species being the most common and harmful causative agent of human malaria worldwide. The success of antimalarial treatments is currently in jeopardy due to the obligate intracellular parasite’s high propensity for drug resistance. New drug targets can be found in an essential chloroplast-like organelle called the apicoplast which contains its own genome that must be replicated and maintained for parasite survival. P. falciparum apicoplast contains a single DNA polymerase (apPOL) that replicates the plastid genome with high fidelity but low processivity and requires the presence of an unidentified apPOL processivity factor to successfully replicate the entire 35 kb genome. Although apPOL genome replication has been investigated, its role within DNA repair pathways is lacking primarily due to a significant underrepresentation of commonly known repair proteins despite recent efforts from both bioinformatics and proteomics. Here, we utilized a combination of biochemical, biophysical, and cell biology-based assays to resolve some of the knowledge gaps associated with apPOL biology, function, and its potential role in drug development. To establish apPOL as an attractive antimalarial target, we investigated small molecule inhibitors against the DNA polymerase and 3’-5’ exonuclease activities of apPOL and tested their strengths in clearing parasites from red blood cell infection. apPOL participation in apicoplast DNA repair of common lesions such as ribonucleotides (rNTPs) was explored by measuring in vitro the rNTP misincorporation frequencies, reverse transcriptase activity, mismatch extension efficiency, and proofreading activity of apPOL on rNTP-containing DNA substrates. Lastly, to elucidate proteins involved with apicoplast DNA repair we generated an inducible C-terminal TurboID fusion on the endogenous apPOL gene to conduct targeted proteomics within cultured parasite. Overall, results from each project suggest apPOL is a suitable drug target that can participate in apicoplast DNA repair of commonly observed DNA lesions. Additionally, highly confident candidates identified through targeted proteomics are essential proteins associated with either apPOL function or apicoplast DNA repair and may serve as future attractive drug targets for antimalarial development. Together this work establishes apPOL as an attractive avenue for small molecule antimalarial development and highlights the important role apPOL plays in parasite survival by contributing to both plastid genome replication and repair activities.