Mycobacterium tuberculosis is an exquisitely adapted intracellular pathogen that
encounters hostile, host-derived reactive nitrogen and oxygen intermediates during
the course of infection of its human host. These radicals cause DNA damage, which
is repaired through various pathways to allow for the continued survival of the
organism. Base excision repair (BER) is one such pathway, which depends on DNA
glycosylases to identify and excise damaged DNA bases. Formamidopyrimidine
DNA glycosylase (Fpg/ MutM/ FAPY) and Endonuclease VIII (Nei) are such
enzymes, which both target oxidatively damaged DNA and together, form the Fpg
family of DNA glycosylases. Bioinformatic analyses identified two copies each of
Fpg and Nei-encoding genes in M. tuberculosis as well as in its non-pathogenic
relative, Mycobacterium smegmatis. To understand the role of these multiple
glycosylases in the maintenance of genomic integrity and survival of mycobacteria,
the genes encoding the four Fpg/Nei glycosylases were individually deleted in M.
smegmatis strain mc2155 by homologous recombination. In addition to the four single
mutants, double and triple Fpg and Nei glycosylase knockout mutants were generated
by sequential gene knockout. When compared to the parental strain, the single and
double mutants showed no variation in growth kinetics, no increased sensitivity to
hydrogen peroxide and no increase in spontaneous mutation rates. However, a slight
increase in frequency of spontaneous C T transition mutations was observed in
double knockout mutants compared to the wild type and single mutant strains. These
results suggest that these enzymes may be part of an extensive network of enzymes
which collectively work to enhance the overall survival of M. smegmatis through the
repair of oxidatively damaged DNA.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/6883 |
| Date | 09 April 2009 |
| Creators | Goosens, Vivianne Jacoba |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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