title: Oxidative damage of nucleic acids &amp; its consequences: towards an understanding at an atomic level
Creators: name: Lambert, Megan Catherine
Publisher: name: University of Reading
Year: 2024

description: 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.