UVA-induced cyclobutane pyrimidine dimers form predominantly at thymine-thymine dipyrimidines and correlate with the mutation spectrum in rodent cells

Abstract

Ligation-mediated PCR was employed to quantify cyclobutane pyrimidine dimer (CPD) formation at nucleotide resolution along exon 2 of the adenine phosphoribosyltransferase (aprt) locus in Chinese hamster ovary (CHO) cells following irradiation with either UVA (340-400 nm), UVB (295-320 nm), UVC (254 nm) or simulated sunlight (SSL; lambda > 295 nm). The resulting DNA damage spectrum for each wavelength region was then aligned with the corresponding mutational spectrum generated previously in the same genetic target. The DNA sequence specificities of CPD formation induced by UVC, UVB or SSL were very similar, i.e., in each case the overall relative proportion of this photoproduct forming at TT, TC, CT and CC sites was approximately 28, approximately 26, approximately 16 and approximately 30%, respectively. Furthermore, a clear correspondence was noted between the precise locations of CPD damage hotspots, and of 'UV signature' mutational hotspots consisting primarily of C-->T and CC-->TT transitions within pyrimidine runs. However, following UVA exposure, in strong contrast to the above situation for UVC, UVB or SSL, CPDs were generated much more frequently at TT sites than at TC, CT or CC sites (57% versus 18, 11 and 14%, respectively). This CPD deposition pattern correlates well with the strikingly high proportion of mutations recovered opposite TT dipyrimidines in UVA- irradiated CHO cells. Our results directly implicate the CPD as a major promutagenic DNA photoproduct induced specifically by UVA in rodent cells.

Figure 1

Induction of CPDs by UVA, UVB, UVC radiation and SSL, at nucleotide resolution, in exon 2 of the aprt gene of CHO cells. Cells were either unirradiated (NoUV) or irradiated as indicated and CPD formation was analyzed by LMPCR along the NTS of aprt gene exon 2. The doses for UVB, SSL and UVC induced approximately equal global CPD frequencies as determined by alkaline denaturing agarose gel electrophoresis of T4 endonuclease V-treated genomic DNA (data not shown). The arrows on the right side of the autoradiograms indicate dipyrimidine sites quantified with phosphorimager. The first four lanes from the left on each autoradiogram exhibit LMPCR of DNA treated in standard Maxam–Gilbert cleavage reactions. (A) The three lanes to the right of the Maxam–Gilbert sequencing ladder represent LMPCR of DNA isolated from cells irradiated with 0.03 kJ/m² of UVC, 0.6 kJ/m² of UVB or 3000 kJ/m² of SSL, respectively. (B) The nine lanes to the right of the Maxam–Gilbert sequencing ladder represent LMPCR of DNA isolated from cells irradiated with (from left to right, respectively): 4500, 3000, 1500 kJ/m² of UVA; 3.75, 1.20, 0.60 kJ/m² of UVB; 7500, 5000, 2500 kJ/m² of SSL.

Figure 1

Induction of CPDs by UVA, UVB, UVC radiation and SSL, at nucleotide resolution, in exon 2 of the aprt gene of CHO cells. Cells were either unirradiated (NoUV) or irradiated as indicated and CPD formation was analyzed by LMPCR along the NTS of aprt gene exon 2. The doses for UVB, SSL and UVC induced approximately equal global CPD frequencies as determined by alkaline denaturing agarose gel electrophoresis of T4 endonuclease V-treated genomic DNA (data not shown). The arrows on the right side of the autoradiograms indicate dipyrimidine sites quantified with phosphorimager. The first four lanes from the left on each autoradiogram exhibit LMPCR of DNA treated in standard Maxam–Gilbert cleavage reactions. (A) The three lanes to the right of the Maxam–Gilbert sequencing ladder represent LMPCR of DNA isolated from cells irradiated with 0.03 kJ/m² of UVC, 0.6 kJ/m² of UVB or 3000 kJ/m² of SSL, respectively. (B) The nine lanes to the right of the Maxam–Gilbert sequencing ladder represent LMPCR of DNA isolated from cells irradiated with (from left to right, respectively): 4500, 3000, 1500 kJ/m² of UVA; 3.75, 1.20, 0.60 kJ/m² of UVB; 7500, 5000, 2500 kJ/m² of SSL.

Figure 2

Distribution of CPDs and mutations induced by UVA, UVB, UVC radiation and SSL in exon 2 of the aprt gene of CHO cells. CPDs were mapped along the NTS of exon 2. Quantification of CPDs was performed as described in Materials and Methods. The upper part of the figure represents the relative frequencies of CPD formation at every dipyrimidine site. In the lower part of the figure, mutations as reported by Drobetsky et al. (13), and mapped along the sequence independently of the strand where the premutagenic lesions at the origin of the base changes are expected. The relative frequency of mutations at each site was calculated as the ratio of the number of mutants recovered at a particular site to the total number of mutants occurring along exon 2 of the aprt gene after irradiation. All events were considered in the calculation and double mutations were counted as one change at each of the two positions. The underlined nucleotides correspond to positions 238–246.

Figure 3

Distribution of CPDs induced by UVA, UVB, UVC radiation and SSL in exon 2 of the aprt gene of CHO cells. CPDs were mapped along the TS. Quantification of CPDs was performed as described in Materials and Methods.