Studies of DNA repair by Cyclobutane pyrimidine dimers

Ladebeck, Oliver 2010. Studies of DNA repair by Cyclobutane pyrimidine dimers. PhD Thesis, Cardiff University.

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Abstract

Cyclobutane pyrimidine dimers (CPDs) are the major photo products that can occur in DNA after UV-light exposure. Detrimental effects of CPDs for organisms can be prevented by the DNA repair enzyme DNA photolyase. This protein repairs CPDs by a light induced (300–500 nm) electron transfer involving a non-covalently bound cofactor, FAD. The key role of FAD in the catalytic DNA repair can be investigated by replacing FAD in the enzyme with FAD analogues and compare the activity of wild-type enzyme to the mutant enzyme. As a part of this study, a stable expression system of photolyase was created and the enzyme was purified in concentrations in the uM range. The FAD analogue roseoFAD was biochemically synthesised from its precursor form roseoflavin. FAD was extracted from photolyase by unfolding the enzyme using the effects of denaturing agents or changes in buffer pH values. The protein was successfully refolded in the presence of roseoFAD and its integration into the proteins active site was confirmed by UV/Vis spectroscopy. Surface plasmon resonance spectroscopy (SPR) was used to monitor DNA repair by photolyase in real-time. For this purpose photolyase was overexpressed and purified. CPDs, the substrate of photolyase, were produced by UV irradiation of poly-T oligonucleotides. SPR signals were detected for steps of the repair process such as enzyme-substrate complex formation, DNA repair and release of product. Changes in SPR signals were used to obtain the kinetics of DNA repair by measuring the on and & off rates and calculating the rate constants for substrate binding and product release. According to the data obtained the dissociation constant KD was calculated and found to be in good agreement with published values obtained by different methods.

Item Type:	Thesis (PhD)
Status:	Unpublished
Schools:	Chemistry
Subjects:	Q Science > QD Chemistry
ISBN:	9781303217913
Date of First Compliant Deposit:	30 March 2016
Last Modified:	19 Mar 2016 23:31
URI:	https://orca.cardiff.ac.uk/id/eprint/54956

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