Characterizing the role of DNA polymerase gamma in the yeast, Cryptococcus neoformans

WCU Author/Contributor (non-WCU co-authors, if there are any, appear on document)
Joshua Ellis Boggs (Creator)
Western Carolina University (WCU )
Web Site:
Indrani Bose

Abstract: The basidiomycetous yeast, Cryptococcus neoformans, is an opportunistic pathogen that is responsible for a common fungal infection of the central nervous system, cryptococcal meningoencephalitis. This is a signal disease of AIDs patients that can also be found in patients of other immune compromising afflictions. This disease can be treated with various antifungal medications, but these drugs must be taken in combination, and are often far too expensive for widespread availability. The current shortcomings of available treatments make it critical that we understand vital systems within this organism that may lead to development of more effective and potentially cheaper medications. DNA polymerase gamma (polG or Mip1p) has been identified as the only polymerase in yeast that is capable of replication and repair of the entire mitochondrial (mt) genome. In S. cerevisiae, deletion of DNA polG leads to the overall lack of mtDNA and the production of petite or wee colonies. C. neoformans is known to be aerobic, and therefore, the integrity of its mitochondrial DNA is hypothesized to be vital to the survival of the yeast. This necessity of mtDNA replication and maintenance makes polG a strong candidate as a potential drug target. Using amino acid sequence homology, a MIP1 homolog has been identified in C. neoformans. This gene, CNAG_06769, has a 45% identity to the MIP1 gene in S. cerevisiae and contains a mitochondrial localization signal (MLS) as well as a DNA polymerase A domain, which is found in all known DNA polG proteins. The polymerase domain identified in C. neoformans polG contains a 62% identity and a 76% similarity to the same domain found in S. cerevisiae polG. There are also regions within the cryptococcal protein that have previously not been seen in other fungal or mammalian polG proteins. This has raised questions regarding what the functions of these domains may be and how these functions may be relevant in DNA polG research. In this project, RNAi has been used to silence the MIP1 gene (MIP1i). Galactose-dependent knock-down of CnMIP1 shows that this gene appears to be essential to C. neoformans survival. Growth curves of the MIP1i strains compared to those of wild type cells show that the strains are unable to proliferate in the absence of this protein. In addition, mitochondrial integrity assays using the vital dye Mitotracker Green indicate that RNAi of MIP1 affects the viability of these cells. Localization of the CnpolG protein with a C-terminal mCherry tag was also attempted. Constructs were created to express this protein from its endogenous promoter, as well as from a copper-repressible promoter. However, the attempts to localize this protein in vivo have been inconclusive to date and require further optimization.

Additional Information

Language: English
Date: 2017
Boggs, Bose, Cryptococcus, MIP1, polG, Polymerase

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