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No description available.

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No description available.

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.

No description available.

DNA repair mechanisms constitute an essential cellular response to DNA damage arising either from metabolic processes or from environmental sources such as ultraviolet radiation. Repair of these lesions may be via direct reversal, or by processes such as nucleotide excision repair (NER), a coordinated pathway in which lesions and the surrounding nucleotides are excised and replaced via DNA resynthesis. The importance of repair is illustrated by human disease states such as xeroderma pigmentosum and Cockayne's syndrome which result from defects in the NER system arising from mutations in XP- genes or XP- and CS- genes respectively Little detail is known of DNA damage repair processes in plants, despite the economic and ecological importance of these organisms. This study aimed to expand our knowledge of the process of NER in plants, largely via a polymerase chain reaction (PCR)-based approach involving amplification, cloning and characterisation of plant genomic DNA and cDNA. Homologues of the NER components XPF/RAD1 and XPD/RAD3 were isolated as both genomic and complete cDNA sequences from the model dicotyledonous plant Arabidopsis thaliana. The sequence of the 3'-untranslated region of atXPD was also determined. Comparison of genomic and cDNA sequences allowed a detailed analysis of gene structures, including details of intron/exon processing. Variable transcript processing to produce three distinct transcripts was found in the case of atXPF. In an attempt to validate the proposed homologous function of these cDNAs, assays to test complementation of resistance to ultraviolet radiation in the relevant yeast mutants were performed. Despite extensive amino acid sequence conservation, neither plant cDNA was able to restore UV-resistance. As the yeast RAD3 gene product is also involved in vivo in transcription, and so is required for viability, the atXPD cDNA was tested in a complementation assay for this function in an appropriate yeast mutant. The plant cDNA was found to substantially increase the viability of the yeast mutant. The structural and functional significance of these results is discussed comparatively with reference to yeast, human and other known homologues. Other putative NER homologues were identified in A. thaliana database sequences, including those of ERCC1/RAD10 and XPG/ERCC5/RAD2, and are now the subjects of ongoing investigations. This study also describes preliminary investigations of putative REVS and RAD30 translesion synthesis genes from A. thaliana.

Graduation date: 2003

Thesis (M.S. in biochemistry)--Washington State University, December 2009. / Title from PDF title page (viewed on Jan. 20, 2010). "School of Molecular Biosciences." Includes bibliographical references.

Thesis (Ph. D.)--University of Oregon, 2001. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 99-114). Also available for download via the World Wide Web; free to University of Oregon users.