The construction and phenotypic characterization of mycobacterial mutants deficient in DNA glycosylases
Year: 2009
Type: dissertation
Abstract: Mycobacterium tuberculosis is an exquisitely adapted intracellular pathogen that \nencounters hostile, host-derived reactive nitrogen and oxygen intermediates during \nthe course of infection of its human host. These radicals cause DNA damage, which \nis repaired through various pathways to allow for the continued survival of the \norganism. Base excision repair (BER) is one such pathway, which depends on DNA \nglycosylases to identify and excise damaged DNA bases. Formamidopyrimidine \nDNA glycosylase (Fpg/ MutM/ FAPY) and Endonuclease VIII (Nei) are such \nenzymes, which both target oxidatively damaged DNA and together, form the Fpg \nfamily of DNA glycosylases. Bioinformatic analyses identified two copies each of \nFpg and Nei-encoding genes in M. tuberculosis as well as in its non-pathogenic \nrelative, Mycobacterium smegmatis. To understand the role of these multiple \nglycosylases in the maintenance of genomic integrity and survival of mycobacteria, \nthe genes encoding the four Fpg/Nei glycosylases were individually deleted in M. \nsmegmatis strain mc2155 by homologous recombination. In addition to the four single \nmutants, double and triple Fpg and Nei glycosylase knockout mutants were generated \nby sequential gene knockout. When compared to the parental strain, the single and \ndouble mutants showed no variation in growth kinetics, no increased sensitivity to \nhydrogen peroxide and no increase in spontaneous mutation rates. However, a slight \nincrease in frequency of spontaneous C T transition mutations was observed in \ndouble knockout mutants compared to the wild type and single mutant strains. These \nresults suggest that these enzymes may be part of an extensive network of enzymes \nwhich collectively work to enhance the overall survival of M. smegmatis through the \nrepair of oxidatively damaged DNA.
Author: Vivianne J. Goosens
Language: English
Subfield: Infectious Diseases
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2161458480