Mutation spectrum in UVB‐exposed skin epidermis of Xpa‐knockout mice: Frequent recovery of triplet mutations

Abstract

Knockout mutations in both alleles of the Xpa gene give rise to a complete deficiency in nucleotide excision repair (NER) in mammalian cells. We used transgenic mice harboring the lambda-phage-based lacZ mutational reporter gene to study the effect of Xpa null mutation (Xpa(-/-)) on damage induction, repair, and mutagenesis in mouse skin epidermis after UVB irradiation. UVB induced equal amounts of cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (64PPs) in mouse skin epidermis of Xpa(-/-) and wild-type mice. Neither photolesion was removed in the Xpa(-/-) epidermis by 12 hr after irradiation whereas removal of 64PPs was observed in the epidermis of wild-type mice. Irradiation with 200 and 300 J/m(2) UVB increased the lacZ mutant frequency in the epidermis of Xpa(-/-) mice, but the induced mutant frequencies were not significantly different from those previously determined for wild-type mice. One-hundred lacZ mutants isolated from the UVB-exposed epidermis of Xpa(-/-) mice were analyzed and compared with mutant sequences previously determined for irradiated wild-type mice. The distribution of the mutations along the lacZ transgene and the preferred dipyrimidine context of the UV-specific mutations were similar in mutants from the Xpa(-/-) and wild-type mice. The spectra of the mutations in the two genotypes were both highly UV-specific and similar in a dominance of C --> T transitions at dipyrimidine sites; however, Xpa(-/-) mice had a higher frequency than wild-type mice of two-base tandem substitutions, including CC --> TT mutations, three-base tandem mutations and double base substitutions that were separated by one unchanged base in a three-base sequence (alternating mutations). These tandem/alternating mutations included a remarkably large number of triplet mutations, a recently reported, novel type of UV-specific mutation, characterized by multiple base substitutions or frameshifts within a three-nucleotide sequence containing a dipyrimidine. We conclude that the triplet mutation is a UV-specific mutation that preferably occurs in NER-deficient genetic backgrounds.

Impaired spermatogenesis and elevated spontaneous tumorigenesis in xeroderma pigmentosum group A gene (Xpa)-deficient mice

“…Finally, in view of our previous observation that Xpa (−/−) mice had an increased frequency of UVB-induced CC-TT mutations (4, 5), it is interesting to note that Reid and Loeb (51) showed that CC->TT mutations are a signature for oxidative stress, perhaps resulting from hydroxyl radical induced cytosine crosslinks. Consistent with this idea, it has been shown that exposure of DNA to oxygen radicals generates DNA lesions at tandem cytosines, as well as tandem purines, that are substrates for the bacterial NER system (52), and presumably for mammalian NER as well.…”

“…We have reported that Xpa (−/−) mice are deficient in the repair of UV-damage and show a higher incidence of UVB-induced skin tumorigenesis and mutation rate in UVB-irradiated skin than Xpa (+/+) mice (2, 4, 5). In the present work, we examined whether spontaneous tumorigenesis is increased in Xpa (−/−) mice.…”

“…Further, three of these cell lines were deficient in UV-induced G1/S cell cycle checkpoint and mismatch repair activity, suggesting that a mismatch repair-defect is involved in the UVB-induced skin carcinogenesis in Xpa (−/−) mice (3, and our unpublished data). Using Xpa (−/−) mice containing the rpsL -reporter gene, we found that the frequency of UVB-induced mutations such as the CC to TT tandem transition was higher than in wild-type mice (4, 5). We also found that UVB-induced immunosuppression was enhanced in Xpa (−/−) mice (6).…”

Analyses of 8-Methyldeoxyadenosine and 8-Methyldeoxyguanosine as Markers of Free Radical-mediated DNA Methylation in Mouse

“…Epidermis DNA extraction: DNA extraction from skin was performed according to the protocol reported by Ikehata et al (13).…”

BAX and Tumor Suppressor TRP53 Are Important in Regulating Mutagenesis in Spermatogenic Cells in Mice1

“…Repair of DNA plays a large role in regulating mutant frequency [4][5][6]. In particular, the base excision repair (BER) pathway plays a major role in regulating mutant frequency in the rodent male germline [7,8].…”