Characterization of DNA polymerase gamma in the basidiomycetous yeast, Cryptococcus neoformans

WCU Author/Contributor (non-WCU co-authors, if there are any, appear on document)
Samuel Richardson Walter (Creator)
Institution
Western Carolina University (WCU )
Web Site: http://library.wcu.edu/
Advisor
Indrani Bose

Abstract: Cryptococcus neoformans is a common basidiomycetous yeast and an obligate aerobe thatinhabits most environments across the world. C. neoformans is also an opportunistic pathogencapable of causing a fungal infection of the central nervous system known as cryptococcalmeningoencephalitis. This disease is often observed in patients suffering from AIDS or otherimmune-compromising afflictions. The antifungal medications currently used to treat C. neoformans infections are expensive and often incapable of eradicating infection. Understanding vital systems in this organism are critical to the production of cheaper and more effectivemedications. Mitochondria are a critically important organelle in aerobic organisms, generating more than 90% of cellular ATP. DNA polymerase gamma (PolG) is a nuclear-encoded DNApolymerase that has been shown in many organisms like humans, Drosophila, and Saccharomyces, to play a vital role in maintaining and replicating mitochondrial DNA. Homology searches have revealed that C. neoformans contains a single putative DNA polymerase gamma gene (CnMIP1). The aims of this project were to determine the essentiality of this gene, and to characterize the biochemical function of the encoded protein (CnPolG). In order to determine if CnMIP1 was essential to the survival of this yeast, the gene was knocked down in vivo using RNA interference. This showed that cryptococci are incapable of survival without CnPolG, thus providing evidence of the importance of CnMIP1 in C. neoformansviability. Although DNA sequence homology showed the presence of a polymerase and an exonuclease domain in CnMIP1, the function of the encoded protein had to be experimentally determined. For this, the CnMIP1 gene was cloned and recombinant protein was expressed in both bacterial (pET28 plasmid) and yeast (pRS424 plasmid) expression systems. The bacteriallyexpressed protein was heavily degraded, but addition of ethanol to the media has shown promising results. The yeast-produced protein, using a codon-optimized gene construct, showed significantly reduced levels of degradation, but this has been an unreliable expression system for large-scale production of this protein thus far. The polymerization ability of recombinant CnPolG purified from bacteria has been assayed using M13 ssDNA as template. Additionally, it has been shown that this protein can utilize either tailed or untailed primers to initiate polymerization and performs more robustly in a low salt environment. Finally, these assays have also demonstrated that polymerase activity is dependent on Mg2+ or Mn2+ ions and that functionality increases when both cations are present. Comparing homologues fromSaccharomyces, humans, and Cryptococcus shows the presence of two domains in CnPolG that are not present in the other two homologues. Moving forward, these domains could provide functional insights into the regulation of mitochondrial polymerases and may serve as potential pharmaceutical targets if they play an essential role in CnMIP1 functionality.

Additional Information

Publication
Thesis
Language: English
Date: 2020
Keywords
Cryptococcus neoformans, DNA Polymerase Gamma, Protein characterization

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