Formation of the Main UV-induced Thymine Dimeric Lesions within Isolated and Cellular DNA as Measured by High Performance Liquid Chromatography-Tandem Mass Spectrometry

Thierry Douki,Magali Court,Sylvie Sauvaigo,Francette Odin,Jean Cadet

doi:10.1074/jbc.275.16.11678

Abstract

UVB radiation-induced formation of dimeric photoproducts at bipyrimidine sites within DNA has been unambiguously associated with the lethal and mutagenic properties of sunlight. The main lesions include the cyclobutane pyrimidine dimers and the pyrimidine (6-4) pyrimidone adducts. The latter compounds have been shown in model systems to be converted into their Dewar valence isomers upon exposure to UVB light. A new direct assay, based on the use of liquid chromatography coupled to tandem mass spectrometry, is now available to simultaneously detect each of the thymine photoproducts. It was applied to the determination of the yields of formation of the thymine lesions within both isolated and cellular DNA exposed to either UVC or UVB radiation. The cis-syn cyclobutane thymine dimer was found to be the major photoproduct within cellular DNA, whereas the related (6-4) adduct was produced in an approximately 8-fold lower yield. Interestingly, the corresponding Dewar valence isomer could not be detected upon exposure of human cells to biologically relevant doses of UVB radiation.

Highlights

Ultraviolet radiation represents the most deleterious part of solar light to cells and has been associated with the occurrence of skin cancer [1]
HPLC-MS/MS Analysis of TpT Photoproducts—Electrospray ionization (ESI) mass spectrometry is a suitable technique for the characterization and the measurement of a wide range of molecules [36]
TpT photoproducts can be analyzed as negative ions. This is a major advantage in a method involving the coupling of HPLC to mass spectrometry

Summary

Introduction

Ultraviolet radiation represents the most deleterious part of solar light to cells and has been associated with the occurrence of skin cancer [1]. The main advantage of the latter assay is that the DNA photoproducts arising from the different bipyrimidine sites can be individually measured with a high specificity depending on the detection system used. This approach has been applied to the quantitation of thymine cyclobutane dimer within isolated [26] and radiolabeled cellular [27, 28] DNA following hot formic acid treatment. The assay was first applied to the measurement of the level of

Methods

Results

Conclusion