Mismatch repair in T. brucei: roles in protection against oxidative damage

Fatima, Tehseen Zeb (2013) Mismatch repair in T. brucei: roles in protection against oxidative damage. PhD thesis, University of Glasgow.

Full text available as:
[thumbnail of 2013FatimaPhD.pdf] PDF
Download (8MB)
Printed Thesis Information: https://eleanor.lib.gla.ac.uk/record=b2987413

Abstract

Cells are continuously exposed to different intracellular and extracellular mutagens, which can damage several different molecules, including DNA. To ensure survival, cells have evolved various defence and repair mechanisms. Mismatch repair (MMR) is the mechanism that serves to repair mismatched bases in DNA that are missed by the proof reading activity of DNA polymerases. Besides this, MMR also corrects base mismatches formed by altered bases modified by certain chemical mutagens. Thus, MMR is important to avoid mutagenesis and maintain genome fidelity. MMR is a complex, highly conserved pathway that involves a range of proteins along with several accessory proteins.
In T. brucei, as in other eukaryotes, MMR core functions are carried out by bacterial MutS and MutL homologues, working as heterodimers: MSH2α (MSH2-MSH3) and MSH2β (MSH2-MSH6), and MLH1-PMS1, respectively. To date, only MSH2 and MLH1 function have been examined and only in bloodstream form (BSF) T. brucei cells. It was observed that MMR mutants are tolerant towards methylation damage, exhibit microsatellite instability and display elevated rates of homologous recombination between imperfectly matched DNA molecules. This confirmed the role of MMR in genome maintenance in BSF T. brucei. More recently, it was observed that BSF MSH2 mutants were sensitive towards oxidative damage, though the same phenotype was not observed in MLH1 mutants. This suggests that some aspect of the MMR machinery acts to protect BSF T. brucei cells against oxidative stress, but the machinery and mode of action is unknown.
In this study we have generated null mutants of MSH2 and MLH1 in procyclic form (PCF) T. brucei cells, and MSH3 and MSH6 mutants in BSF cells. Characterization of tolerance to DNA methylation damage and evaluating microsatellite stability shows that each gene acts in MMR in both the life cycle stages, with the exception of MSH3, where null mutants show no discernible phenotypes. Mutants were also analyzed for their action towards oxidative stress in both the life stages and, remarkably, we find life cycle stage differences, with MSH2 mutants displaying hydrogen peroxide sensitivity and resistance in the BSF and PCF, respectively. The same phenotypes are not seen in MLH1 mutants, and we show that resistance to hydrogen peroxide in PCF cells is due to adaptation during the loss of MSH2. We have also shown that PCF MSH2 mutants may show a decrease in microsatellite instability when subjected to oxidative stress. This leads to the hypothesis that there might be an unidentified system, apart from MMR, present in T. brucei PCF cells that works as a defence in response to oxidative stress and can assume greater prominence when MSH2 is lost. Although we have tried to explore various cellular processes that might contribute this activity, our results are inconclusive.
MSH2 and MLH1 have also been epitope tagged to explore the subcellular localization of these proteins and to ask if any changes in expression levels or changes in localisation are seen when subjected to oxidative stress. These preliminary data suggest that both factors are nuclear and cytoplasmic. We have also tried to ask if MSH2 and/or MLH1 co-localize with either MSH5 or MSH4, which are MutS-like factors that act in meiosis in other eukaryotes, but whose functions have not been explored in T. brucei. However, our attempts at this analysis have been unsuccessful.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: T. brucei, Oxidative damage, mismatch repair
Subjects: Q Science > QR Microbiology > QR180 Immunology
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Parasitology
Supervisor's Name: McCulloch, Dr. Richard
Date of Award: 2013
Depositing User: Mrs. Tehseen Fatima
Unique ID: glathesis:2013-4554
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 29 Aug 2013 10:51
Last Modified: 29 Aug 2016 13:25
URI: https://theses.gla.ac.uk/id/eprint/4554

Actions (login required)

View Item View Item

Downloads

Downloads per month over past year