Enzymatic recognition and biological effects of photodynamic damage induced in DNA by 1,6-dioxapyrene plus UVA.

The specific recognition of DNA modifications by repair endonucleases was used to characterize DNA damage induced by 1,6-dioxapyrene (1,6-DP) in the presence of ultraviolet light at 365 nm (UVA) in the plasmid YEplac181. Under cell free conditions, 1,6-DP plus UVA generated lesions are recognized by the UvrABC endonuclease, the proteins Nth, Nfo and Fpg. The number of UvrABC sensitive sites was at least ten-fold higher than that of Fpg or Nth sensitive sites. Moreover, 1,6-DP plus UVA generated single-strand breaks which are the second most frequent lesions. To investigate the biological effect of DNA damage, YEplac181 DNA was treated with 1,6-DP plus UVA and transformed into Escherichia coli or Saccharomyces cerevisiae. In Escherichia coli, the transformation efficiency of 1,6-DP plus UVA treated DNA was greatly reduced in the uvrA mutant compared to that in the wild-type strain. However, the transforming efficiency was not affected in Fpg-deficient strains. In Saccharomyces cerevisiae, the transformation efficiency of 1,6-DP plus UVA treated YEplac181 was greatly reduced in the rad14::URA3 strain. The photobiological effect of 1,6-DP plus UVA was also analysed in haploid yeast strains of various repair capacities. The results show that the yeast strain defective in the nucleotide excision repair pathway (rad14::URA3) is hypersensitive to 1,6-DP plus UVA treatment as compared to the parental wild-type strain. It is confirmed that the lethal effect of 1,6-DP plus UVA on wild-type yeast is strongly oxygen dependent, whereas the survival of the rad14::URA3 mutant only exhibits a minor oxygen dependence. To conclude, our data show that the photodynamic DNA lesions induced by 1,6-DP plus UVA can be recognized and repaired in pro- and eukaryotic cells by the nucleotide excision repair pathway.