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Title: A novel mechanism regulating DNA-damage repair in Mycobacterium tuberculosis
Author: Dawson, Lisa Frances
ISNI:       0000 0001 3418 7131
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2006
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The intracellular pathogen Mycobacterium tuberculosis (M. tuberculosis) resides in macrophages and is the causative agent of human tuberculosis. Infected macrophages produce reactive oxygen and nitrogen intermediates, known to damage DNA therefore DNA damage repair is thought to be important in survival of M. tuberculosis in the host. The expression of many bacterial DNA repair genes is often regulated by the SOS response, in which RecA is an integral part however, in M. tuberculosis the majority of genes in the DNA-damage regulon are regulated independently of the RecA/LexA system. In this study two potential mechanisms for this alternative mode of gene regulation were investigated. The first hypothesis addressed was that regulation of expression following DNA-damage is controlled by an alternative sigma factor. Sigma factors are protein subunits of RNA polymerase, which confer specificity of binding to particular promoters. The function/expression of alternative sigma factors is usually regulated by various mechanisms. The sigma factor SigG is the most highly induced of all 13 sigma factors of M. tuberculosis in response to DNA-damage in both wild-type and ArecA strains. A knockout of sigG in M. tuberculosis was constructed, and found to be more susceptible to mitomycin C stress than wild-type H37Rv and attenuated in mice. ruvC was shown to possesses 2 transcriptional start sites, and although neither were regulated by SigG, the PI promoter appeared to be dual regulated by LexA and the RecA/LexA independent mechanism. Microarray analysis revealed that SigG was not significantly involved in regulation of the RecA/LexA independent DNA-damage regulon, but that SigG directly or indirectly regulated expression of 127 genes in the absence of DNA-damage. The other possible mode of RecA/LexA independent regulation was via a repressor/activator protein. Gel shifts assays using M. tuberculosis cell free extracts were used to attempt to identify a repressor or activator protein that bound to the operator of the recA PI promoter, known to be induced independently of RecA, but failed to detect specific binding. Published microarray data revealed that Rv2017, a predicted regulatory protein, was upregulated in both wild-type and ArecA strains of M. tuberculosis in response to DNA-damage. Therefore, a gene inactivation knockout of Rv2017 was constructed and analysed in M. tuberculosis. The ARv2017 strain was hyposensitive to mitomycin C stress and preliminary mouse in-vivo infection data suggested that the ARv2017 strain may be hypervirulent. Microarray data revealed that Rv2017 plays a direct/indirect role in regulation of a large regulon, including some genes in the DNA-damage repair regulon.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available