Kershaw, Rachael Maria (2011)
Ph.D. thesis, University of Birmingham.
Reactive oxygen species (ROS) are generated via endogenous and exogenous sources. ROS are involved in essential cellular processes but when present in excess, can overwhelm antioxidant defences and induce a range of damaging DNA lesions. The most commonly oxidised DNA base is guanine. This generates products including 7,8-dihydro-8-oxodeoxyguanine (8-oxo dG) which can result in G:C->T:A transversion mutations, frequently found in human cancers. Oxidative damage is also implicated in normal cellular ageing and degenerative diseases. 8-oxo dG repair is initiated by the base excision repair enzyme 8-oxoguanine DNA glycosylase 1 (OGG1). Modulation of OGG1 activity in oxidising conditions has implications for mutation prevention. This thesis investigates regulation of OGG1 under oxidising conditions using BSO, which increases intracellular ROS. Chapter 3 shows that following BSO treatment, mouse OGG1 activity in mouse embryonic fibroblast (MEF) cells increases with no change in mRNA levels, whereas identical treatment has no effect on rat OGG1 activity in MH1C1 cells but modulates protein levels. A human OGG1 (hOGG1) variant with serine exchanged for cysteine at codon 326 (Cys326-hOGG1) is associated with reduced repair ability under oxidising conditions. Chapter 4 describes the development of mOGG1-/- MEF cells stably expressing Ser326- or Cys326-hOGG1 and in chapter 5 these cells are used to investigate Ser326- and Cys326-hOGG1 activity, gene expression, protein localisation, homo-dimer formation and retention within an insoluble nuclear fraction following BSO treatment. Data presented shows that the activity of both Ser326- and Cys326-hOGG1 increase following BSO treatment but Ser326-hOGG1 peak activity occurs 12 hours prior to that of Cys326-hOGG1. This increased activity is not associated with increased gene expression or protein, or any protein localisation change; however, Cys326-hOGG1 is retained to a lesser extent in an insoluble nuclear fraction following BSO treatment. The findings presented in this thesis show that OGG1 activity is modulated post-transcriptionally in response to increased ROS and provide a possible mechanism behind impaired Cys326-hOGG1 repair in oxidising conditions, further supporting the role of Cys326-hOGG1 in the process of carcinogenesis.
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