| Summary: | Single cell gel electrophoresis (SCGE), more commonly known as the Comet assay, is
an uncomplicated, affordable and versatile method for investigating DNA damage and
repair. Existing comet–assay based methods were modified and applied in this study in
order to examine the effects of different perturbations on the DNA repair capacity of
different samples.
Mitochondrial functioning has a vast effect on overall cell physiology and does not
simply involve the production of energy in the form of ATP that sustains common
biological processes, but is also associated with important cellular occurrences such as
apoptosis and ROS production. It is suggested that a change in mitochondrial function
may lead to extensive ROS production which may negatively affect macromolecules,
including proteins involved in DNA repair pathways, and impaired energy formation
which in turn may hamper the proper occurrence of energy driven processes. Complex I
and ?III knock–down systems established in 143B cells are used to investigate the effect
that perturbations of the energy metabolism may have on DNA repair capacity.
Metallothioneins (MTs) are known to play an imperative role in trace element
homeostasis and detoxification of metals and are effective ROS scavengers. The prooxidant
environment that heavy metal imbalance causes may result in mutagenesis and
transformation through DNA damage. It is suggested that an imbalance in the metal
homeostasis caused by MT knock–out may create an environment favourable for DNA
damage formation and at the same time impair DNA repair pathways. Because of the
multi–functionality and involvement of metallothioneins in such a wide variety of
biological processes, it was considered interesting and essential to extend the
investigation on the effect of the absence of metallothioneins on DNA repair. A
metallothionein I and ?II knock–out mouse model is employed to determine the effect of
MT knock–out on DNA repair capacity.
It was clear from the results obtained that transfection of cells, as used to investigate a
perturbation in the energy metabolism in 143B cells, has an impairing effect on DRC. It
was also confirmed that metallothioneins play an important and diverse role in cell
biology since the absence thereof inhibits both BER and NER. === Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.
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