Biochemical Characterization of the DNA Helicases RecG, XPB and DinG in Mycobacterium tuberculosis
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AbstractMycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis, a deadly human disease that kills millions of people every year. Mtb is notable for its survival and proliferation inside the harsh environment of the human macrophage, where many bacterial pathogens normally perish. This pathogen has an efficient DNA repair system that helps to maintain the integrity of its genome despite the severe genotoxic stress inside the macrophage, thereby enabling its intracellular survival and proliferation. Helicases, motor enzymes that unwind duplex nucleic acid strands into single strands, are vital enzymes required in virtually every aspect of nucleic acid metabolism, including DNA replication, repair, recombination and transcription. In this thesis, biochemical characterization of three Mycobacterium tuberculosis helicases; namely RecG, XPB and DinG is reported. This study specifically focussed on examining the DNA substrate specificity, ATPase activity, strand annealing activity, and nucleoside triphosphate and divalent metal preferences of these helicases. A number of site-specific mutations in various motifs of RecG was also constructed and their effects assessed. The study also examined the relative expression level of the genes encoding RecG, XPB and DinG helicases under a variety of genotoxic stress conditions. The studies presented in this thesis provide important insights into the function and possible roles of these helicases in M. tuberculosis and facilitate subsequent attempts to delineate the precise roles of RecG, XPB and DinG in Mtb genome maintenance and cellular fitness.
List of papers
|Paper I: Mycobacterium tuberculosis RecG Binds and Unwinds Model DNA Substrates with a Preference for Holliday Junctions. Ephrem Debebe Zegeye, Seetha V. Balasingham, Jon K. Lærdahl, Håvard Homberset and Tone Tønjum (2012). Microbiology 158 (8): 1982-93. This is an open-access article distributed under the terms of the Creative Commons Attribution License. The published version of this paper is available at: https://doi.org/10.1099/mic.0.058693-0|
|Paper II. Effects of conserved residues and naturally occurring mutations on Mycobacterium tuberculosis RecG helicase activity. Ephrem Debebe Zegeye, Seetha V. Balasingham, Jon K. Lærdahl, Håvard Homberset, Mariann Nilsen and Tone Tønjum. (Manuscript in preparation). The paper is removed from the thesis in DUO due to publisher restrictions.|
|Paper III: Enzymatic Activities and DNA Substrate Specificity of Mycobacterium tuberculosis DNA Helicase XPB. Seetha V. Balasingham, Ephrem Debebe Zegeye, Håvard Homberset, Marie L. Rossi, Jon K. Lærdahl, Vilhelm A. Bohr and Tone Tønjum (2012). PLoS One 7(5): e36960. This is an open-access article distributed under the terms of the Creative Commons Attribution License. The published version of this paper is available at: https://doi.org/10.1371/journal.pone.0036960|
|Paper IV. Seetha V. Balasingham, Ephrem Debebe Zegeye, Håvard Homberset, Mariann Nilsen, Marie L. Rossi, Vilhelm A. Bohr and Tone Tønjum. Mycobacterium tuberculosis DinG helicase exhibits a 3'?5' directionality and DNA strand annealing activity (Submitted to Nucleic Acids Research). The paper is removed from the thesis in DUO due to publisher restrictions.|