Mechanisms of Lesion Induction on DNA by UVA Light and its Biological Effects

Abstract

The purpose of this work is the irradiation of samples of plasmid DNA with UVA light in the presence of different substances to study the possible mechanisms involved in the induction of DNA lesions by these wavelengths, such as: catalase to eliminate H2O2 in the solution; EDTA / Chelex 100 to remove metals that might be bound to the DNA molecule such as iron and copper; the involvement of singlet oxygen with performed irradiation and deuterated water (D2O), and addition of inhibitors such as azide in the solution; nucleotides to the DNA sample to verify a possible absorption of UVA by nitrogenous bases and the consequent formation of excited state, resulting in reactive species inside the DNA molecule; irradiation in water and saline buffers to check if the change caused in the conformation of the DNA molecule leads to a greater or lesser absorption of UV light by the bases.The determination and quantification of different types of DNA lesions formed after irradiation will be performed with the use of enzymes for DNA repair (glycosylase and endonucleases) that recognize and cleave the site containing specific lesions. Complementing these analysis tests with antibodies specific for CPD lesions and 6-4PPs will be conducted . Furthermore, we intend to analyze the biological effects of these lesions in the transcription of reporter genes in human cells, both proficient and deficient in DNA repair. These tests should be performed by determining the luciferase protein activity, encoded by the reporter gene. So with the implementation of these biological and biochemical analysis we seek to uncover how the damages induced by UVA light in the DNA molecule are generated by examining the indirect (photosensitizers) and direct (wavelength absorption by the DNA molecule) mechanisms.