Effects of UV radiation exposure on DNA and DNA repair enzymes

• Author(s): Camacho, Inês Sofia Cortes Eusébio
• Date: 2012
• Persistent ID: http://hdl.handle.net/10362/8263
• Origin: Repositório Institucional da UNL
• Subject(s): Endonuclease III; Plasmid DNA; Protein‐DNA complex; UV radiation; Electrophoretic mobility shift assays; Bioelectrochemistry
• Document Type: Master Thesis
• Language: English
• Advisor(s): Folgosa, Filipe

Description

Dissertação para obtenção do Grau de Mestre em Biotecnologia DNA integrity in the cell is under constant threat from damaging agents of endogenous or exogenous origin, such as UV light, ionizing radiation and oxidative stress. Although the effects of these carcinogens on DNA have been extensively studied, very little is known about their effect on DNA repair enzymes. The aim of the present work was the study of the effect of UV radiation on E. coli Endonuclease III, a DNA glycosylase belonging to base excision repair system. This enzyme was homologously overexpressed and then purified with a Fe/protein ratio of 3.88 ± 0.63 (fully‐loaded form). Endonuclease III exposure to UV radiation for 45 min (19.77 kJ dose) may lead to the destruction of the Fe‐S bonds of the [4Fe‐4S] cluster or to the conversion of this center into a different redox state. Electrophoretic mobility shift assays with protein‐DNA complex showed that Endonuclease III binding to plasmid DNA promotes a retardation of the free supercoiled DNA band, indicative of Endonuclease III‐DNA complex(es) formation. These assays also showed that Endonuclease III is able to bind both linear and supercoiled plasmid DNA, although with higher affinity for the linear form. Electrophoretic mobility shift assays performed after 45 min of UV irradiation (19.77 kJ) revealed that although shift occurred, the complexes formed were unstable and dissociated during electrophoresis. Moreover, the presence of aggregates suggests the unfolding of some Endonuclease III molecules. After 6 h of UV irradiation (158.18 kJ) no complexes are formed, leading to the conclusion that Endonuclease III molecules were irreversibly damaged. The electrochemical studies were performed by cyclic and differential pulse voltammetry techniques, at room temperature and anaerobic conditions; Endonuclease III and Endonuclease IIIDNA complex were adsorbed on a bare pyrolytic graphite electrode. For the first time, the direct electrochemical response of Endonuclease III unbound to DNA was observed, with a quasi‐reversible redox couple displaying a midpoint potential of 178 ± 9 mV vs. NHE. Endonuclease III binding to plasmid DNA promotes a positive shift (19 mV vs. NHE) in the characteristic redox couple of Endo III. Protein‐DNA complex UV irradiation promotes a negative shift in its redox potential of 25 mV vs. NHE.