A thesis submitted to the Faculty of Science, University of the Witwatersrand,
Johannesburg, in fulfillment of the requirements for the degree of Doctor of
Philosophy
February 2013 / Mycobacterium tuberculosis is presented with environmental host assaults that damage its
DNA during infection. Tubercle bacilli possess mechanisms to protect against moststresses
imposed by the host, including genotoxic stress. However, tolerance of DNA lesions that
have escaped the normal repair processes requires the function of specialist DNA
polymerases that can introduce mutations during translesion synthesis (replication by-pass),
thus leading to damage-induced mutagenesis. Mycobacteria employ a novel DNA
polymerase, DnaE2, for DNA damage tolerance and induced mutagenesis. DnaE2 belongs to
the C-family of DNA polymerases, which are known to replicate DNA with high fidelity, and
has been implicated in virulence and the emergence of rifampicin resistance of M.
tuberculosis in vivo. In this study, DnaE2 was shown to function in the same pathway as two
accessory proteins, ImuB and ImuA’, for damage tolerance and induced mutagenesis in
mycobacteria. In this system, DnaE2 performs the polymerase function in translesion
synthesis whereas ImuB is a cryptic Y-family DNA polymerase that lacks critical active site
residues. It contains a β-clamp binding motif that allows interaction with the β-clamp and
presumably enables DnaE2 and ImuA’ to access the replication fork. ImuB has a C-terminal
region extending from the β-clamp binding motif which contains disordered regions that
allow the interaction with other proteins and is important for function. ImuA’ is also essential
for damage tolerance and induced mutagenesis but its function remains unknown. This
protein is structurally similar to Escherichia coli RecA protein in the N-terminus and the
middle domain, but it has a distinct C-terminus that was shown to be important for the
interaction with ImuB. The essential replicative, C-family polymerase, DnaE1, was shown to
be upregulated in response to DNA damage and was also shown to interact with ImuB. To
explore the possibility that other proteins are involved in this pathway, ImuB was Cterminally
tagged for use as bait in pull-down experiments in M. smegmatis. However,
introduction of the tag disrupted ImuB function, further reinforcing the importance of the Cterminal
region of ImuB for the function of this protein, presumably via protein-protein
interactions. In contrast, a variant of ImuA’ which was N-terminally tagged was shown to
retain functionality; however, experiments using this protein as a bait for pull-down proved to
be unsuccessful. Proteomic analysis of wild type M. smegmatis, a dnaE2 deletion mutant and
complemented derivative was carried out on cells exposed to the same conditions as used in
the pull-down assay. Base excision repair (BER) components were identified in this analysis,
but did not detect ImuB and ImuA’, suggesting that the levels of expression of these proteins
were comparatively lower under the conditions tested resulting in failure of the pull-down
experiment. Finally, numerous attempts were made to express and purify recombinant forms
of ImuB and ImuA’ in E. coli for use in structural studies. Both proteins were expressed in
the soluble and insoluble fractions; however the levels of soluble protein were low, and as a
result, purified protein preparations could not be obtained.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/12918 |
| Date | 29 July 2013 |
| Creators | Ndwandwe, Duduzile Edith |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
Page generated in 0.1067 seconds