Rantsi, Tebogo Christina2018-07-062018-07-062017https://hdl.handle.net/10539/24787A thesis submitted to the Faculty of Health Science, University of the Witwatersrand, Johannesburg, in the fulfilment of the requirements of the degree of Master of Science in Medicine. Johannesburg, 2017.During infection, Mycobacterium tuberculosis encounters hostile conditions which result in the generation of host-derived reactive oxygen (ROS) and nitrogen species (RNS) as part of the immune response to control the infection. Exposure to these reactive radicals can lead to oxidative damage of DNA, which ultimately destabilises the genome and introduces mutations. However, M. tuberculosis is well equipped with a number of DNA repair pathways such as the base excision repair (BER) pathway, which plays a role in maintaining genome stability and survival of the pathogen. A number of DNA glycosylases are involved in the BER pathway, including formamidopyrimidine (Fpg), endonuclease VIII (Nei) and endonuclease III (Nth), which are the initial enzymes responsible for recognition and excision of damaged DNA bases. It was previously demonstrated that combinatorial deletion of nth and two nei homologues in Mycobacterium smegmatis resulted in reduced survival under oxidative stress conditions with a corresponding increase in mutation rates, suggestive of interplay between these enzymes. To understand the molecular basis of this interplay, the individual effects of the Nei homologues (NeiI and NeiII), together with Nth on survival and mutagenesis under oxidative stress conditions, expected to induce DNA damage, were investigated in the current study. Two mutants lacking nth and either neiI or neiII were generated by homologous recombination. These double deletion mutants together with the individual deletion mutants, the parental strain and the respective complemented strains were phenotypically characterized under oxidative stress conditions and assessed for increased mutagenesis as measured by rifampicin resistance. Defects in the BER system resulted in reduced survival under oxidative stress conditions. Deletion of nth combined with the neiII homologue led to reduced survival under oxidative stress conditions and an increase in spontaneous mutagenesis to rifampicin when compared to the deletion of nth combined with the neiI homologue. Collectively these data suggest that NeiII may play an important physiological role in BER in comparison to the NeiI homologue.enMycobacterium smegmatisMycobacterium TuberculosisThesis