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

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.Ultraviolet radiation represents the most deleterious part of solar light to cells and has been associated with the occurrence of skin cancer (1). UVB (290 -320 nm) radiation is highly mutagenic (2, 3) and mostly induces mutations at bipyrimidine sites in cellular DNA (4 -7). Interestingly, a similar mutation spectrum was observed in the p53 gene of skin tumors cells (8,9). Altogether, these data outlined the biological role of dimeric photoproducts of pyrimidine DNA bases. In the last four decades, major efforts have been devoted to the isolation and the characterization of bipyrimidine photolesions in model compounds and isolated DNA (for reviews, see Refs. 10 and 11). These include the cis-syn and trans-syn diastereoisomers of cyclobutane dimers and the pyrimidine (6-4) pyrimidone adducts (Fig. 1). The latter lesions have been shown to undergo an efficient photoconversion into their Dewar valence isomers upon exposure to UVB light (12).Information on the rate of formation of each specific photoproduct is still needed. Indeed, most of the assays developed for the detection of UV-induced photoproducts within DNA involve the use of indirect methods that do not allow differentiation, for a given class of photoproduct, of the lesions arising from the different possible bipyrimidinic sequences. Cyclobutane pyrimidine dimers have been extensively detected by using T4 endonuclease V, which exhibits a N-glycosylase activity at the 5Ј-extremity of the lesion (13). This leads to the formation of strand breaks, which can be quantified using electrophoretic techniques. A second DNA repair system, namely the Escherichia coli Uvr ABC complex, has been used in combination with a photolyase for the quantitation of cyclobutane dimers and (6-4) photoproducts (14). Another widely applied biochemical approach involves the use of either polyclonal or monoclonal antibodies able to recognize specific classes of pyrimidine photoproducts (15)(16)(17)(18)(19)(20)(21)...

UVB and UVA radiation-mediated damage to isolated and cellular DNA

“…Thus, this allows complete sep- aration and accurate detection of the 12 possible bipyrimidine adducts at TT, TC, CT, and CC. The high sensitivity of the tandem MS detection technique when used in the multiple reaction monitoring (MRM) mode is illustrated by the precise measurement of the main bipyrimidine photoproducts in isolated and cellular DNA upon exposure to low doses of UVC and UVB radiation [36,37]. Another interesting feature of the MRM detection mode is that it provides a specific way of differentiating CPD from 6-4PP for a given bipyrimidine sequence that exhibits the same molecular weight, taking advantage of the occurrence of different fragmentation patterns [38].…”

“…Initially, the formation of dimeric photoproducts at dithymine sites was determined in the DNA of UVB-irradiated THP1 human monocytes within the dose range (0-600 J m -2 ) [36]. Interestingly, the conditions of low dose exposure that were used were found to minimize the occurrence of secondary photoreactions such as photoreversion of CPDs and photoisomerization of 6-4PPs.…”

Preventive Effect of Zinc Against Cadmium-induced Oxidative Stress in the Rat Testis

“…The DNA pellet was then solubilized into 100 μl of deionised water containing 0.1 mM defer-oxamine mesylate (Sigma). The DNA solution was incubated with 2 units of nuclease P1 (Boehringer Mannheim, Meylan, France), and the sample was held at 37 C for 2 h. Then, 4 units of alkaline phosphatase (Boehringer Mannheim) were added [28]. After incubation for 1 h at 37 C, the samples were centrifuged for 5 min at 5,000 × g, and the aqueous layer was collected and analysed by HPLC-EC.…”

Inhibition of Nucleotide Excision Repair by High Mobility Group Protein HMGA1

“…Among a variety of possible UV-induced photoproducts, cyclobutane pyrimidine dimers (CPDs) are the most abundant, stable forms (17) and, if unrepaired, are known to cause mutations and skin cancer (2,18). CPDs alter DNA structure from normal B-form, causing severe bending of the helical axis and disruption of Watson-Crick base pairs at the lesion sites (19).…”

“…Stable DNA photoproducts produced by UV-induced covalent linkage between adjacent bases are prototypes of helical distorting, bulky lesions (17). Among a variety of possible UV-induced photoproducts, cyclobutane pyrimidine dimers (CPDs) are the most abundant, stable forms (17) and, if unrepaired, are known to cause mutations and skin cancer (2,18).…”