Mechanisms of mutagenesis in Mycobacterium tuberculosis: structural and functional characterisation of the DNA polymerase accessory factors encoded by Rv3394c and Rv3395c

Ndwandwe, Duduzile Edith
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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.
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