Abstract/Summary

During normal cell metabolism, the regulation of reactive oxygen species (ROS) is important to keep levels appropriate to maintain cell function. However, during oxidative stress, when ROS production increases and the cell’s ability to neutralise ROS is diminished, many cellular components can be damaged. Oxidative DNA damage induced by ROS, the formation of which can be catalysed by transition metals, has been reported as frequent in an oxidative stress environment. Transition metals bind to DNA via the phosphate backbone and the endocyclic atoms of nucleic acids e.g. at the N7 atoms of purine bases. They have also been shown to catalyse the formation of reactive oxygen species, including hydroxyl radicals HO•, and may therefore act as hotspots for oxidative damage with nearby bases. It is unknown if catalysis occurring at these spots would damage the closest base or if the bound transition metal would protect the base it is bound to from damage. Using X-ray crystallography, a common technique used to study nucleic-acid structures at an atomic scale, the work presented in this thesis describes the development of a new method to identify oxidative damage hotspots in nucleic-acid structures. The objective is to treat nucleic acid crystals, containing ordered bound metal sites, with hydrogen peroxide to induce oxidative damage. The effects of oxidative damage on nucleic acid structures were also investigated in solution using circular dichroism (CD) spectroscopy. Two approaches are used to add transition metals to crystal systems containing nucleic acids. These are cocrystallisation and soaking - the advantages and disadvantages of these two methods in the context of this work were carefully examined. Soaking was considered as a more suitable approach, as cocrystallisation was found to change the structure and cause crosslinking which reduced transition-metal availability to hydrogen peroxide. Applying this method, the binding preferences of transition metals such as copper and iron were investigated. Two transition metals, copper and iron, were found to bind differently to the same DNA structure, a result that could explain why oxidative damage differs between systems containing copper or iron. Furthermore, the same system was used to soak DNA crystals with hydrogen peroxide for the first time. No damage was identified but ordered hydrogen peroxide molecules bound to copper (II) ions were described, giving insight into how the Fenton reaction might progress when copper (II) is bound to the DNA. Finally, using CD, a G-quadruplex forming sequence present in the promoter region of PSEN2, a gene implicated in Alzheimer’s disease, was found to undergo conformational changes in an oxidative stress environment. The structural effects of the presence of more than four tracts of guanines in the sequence were investigated, using base substitutions to suppress quadruplex formation and mimic oxidative damage. This gave insight into the potential equilibrium between all the potential G-quadruplex topologies in this system and how oxidative damage can influence topological changes. This method is a starting point to examine more sophisticated nucleic-acid systems such as G-quadruplexes. Whilst oxidative damage could not be directly visualised in the crystal structures, the use of microcrystals combined with micro-focused X-ray beams at synchrotrons or XFEL sources may lead to visualisation in the future.