Inter-strand photoproducts are produced in high yield within A-DNA exposed to UVC radiation

Abstract

Far-UV irradiation of DNA leads to the dimerization of pyrimidine bases, resulting in the formation of cyclobutane type dimers and (6-4) photoproducts. In the dry state, an additional thymine dimeric photolesion, the spore photoproduct, is also generated. While most photoproducts are expected to be produced between adjacent pyrimidines, little attention has been paid to lesions involving bases located on different DNA strands. Using HPLC- mass spectrometry analysis of enzymatically digested DNA, we observed that, in the dry state, inter-strand dimeric photoproducts represented 30% of the total yield of dimeric thymine lesions. The major inter-strand damage was found to be the spore photoproduct. Formation of inter-strand lesions in significant yield could be obtained in solution upon modification of the DNA conformation as the result of the addition of large amounts of ethanol. In both cases, DNA is in the A-form, which is characterized by a high compaction, likely to favor inter-strand photoreactions. Since the latter DNA conformation is also predominant in bacterial spores, the formation and repair of dimeric photoproducts involving thymine bases located on different DNA strands may thus be relevant in terms of deleterious effects of UV radiation to the latter microorganisms.

Figure 1

UV-induced thymine dimeric lesions within DNA. (A) Chemical structure of the photoproducts involving adjacent bases. The stereochemistry of the spore photoproduct formed within DNA has not yet been determined. (B) Schematic representation of the different isomers of the thymidine cyclobutane dimer. dR, 2-deoxyribose. cis,syn and trans,anti cyclobutane dimers are meso forms and thus exist as a single isomer.

Figure 2

Principle of the enzymatic and acidic release of inter- and intra-strand photoproducts from DNA. B, normal base; dR, 2-deoxyribose; P, phosphate.

Figure 3

Chromatograms recorded upon analysis of Thd exposed to UVC radiation in (a) frozen aqueous solution, (b) lyophilized aqueous solution and (c) air-dried ethanolic solution. An octadecylsilyl silica gel column (3 µm particle size, 150 × 2 mm i.d.) was used at a temperature of 28°C. The mobile phase (flow rate 200 µl min^–1) was a gradient of acetonitrile (maximum proportion 20% v/v) in 2 mM ammonium formate. The elution was monitored by a UV spectrophotometer set at 260 nm. Other dimeric photoproducts are not detected due to their low absorption at the latter wavelength.

Figure 4

Formation of the spore photoproduct within UV-irradiated freeze-dried DNA. The lesion was quantified as dinucleoside monophosphate (TpT, intra-strand) or thymidine (Thd, inter-strand) dimeric photoproduct in enzymatically digested DNA. The reported level of Thd spore photoproducts represents the sum of the amount of the two diastereoisomers. The total level of spore photoproduct, released as a thymine lesion, was measured in acid-hydrolyzed DNA samples. Results represent the mean ± SD of three independent experiments.

Figure 5

Distribution of intra- and inter-strand thymine photoproducts within UVC-irradiated freeze-dried DNA (<>c,a, <>c,s, <>t,s and <>t,a, cis,anti, cis,syn, trans,syn and trans,anti cyclobutane dimers). Yields of formation of intra-strand photoproducts were taken from Douki and Cadet (26). Results represent the yield of formation ± SE obtained by linear regression of the level of lesion with respect to the applied dose (n = 15). For the trans,syn and cis,anti cyclobutane dimers, the (6–4) and the spore photoproducts, reported values represent the sum of the yield of the two diastereoisomers.

Figure 6

Effect of the presence of ethanol on the yield of formation of intra- and inter-strand spore photoproducts and inter-strand trans,anti thymine cyclobutane dimer. Each point represents, for a defined ethanol concentration, the yield of formation (± SE) obtained by linear regression of the level of lesion with respect to the applied dose (n = 12).

Figure 7

Distribution of inter-strand lesions and spore photoproducts within isolated DNA exposed to UVC light in aqueous solution containing 80% ethanol. Each value represents the yield of formation (± SE) obtained by linear regression of the level of lesion with respect to the applied dose (n = 12).