Padula M, Averbeck S, Boiteux S, Averbeck D
Département de Radiobiologie et Radiopathologie, UMR217 Centre National de la Recherche Scientifique, Fontenay aux Roses, France.
J Photochem Photobiol B. 1997 Nov;41(1-2):60-6. doi: 10.1016/s1011-1344(97)00082-1.
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.
通过修复核酸内切酶对DNA修饰的特异性识别，来表征质粒YEplac181在365 nm紫外线（UVA）存在下，由1,6 - 二氧杂芘（1,6 - DP）诱导的DNA损伤。在无细胞条件下，1,6 - DP加UVA产生的损伤可被UvrABC核酸内切酶、Nth、Nfo和Fpg蛋白识别。UvrABC敏感位点的数量比Fpg或Nth敏感位点至少高十倍。此外，1,6 - DP加UVA产生的单链断裂是第二常见的损伤。为了研究DNA损伤的生物学效应，用1,6 - DP加UVA处理YEplac181 DNA，并将其转化到大肠杆菌或酿酒酵母中。在大肠杆菌中，与野生型菌株相比，uvrA突变体中1,6 - DP加UVA处理的DNA的转化效率大大降低。然而，在Fpg缺陷菌株中转化效率不受影响。在酿酒酵母中，1,6 - DP加UVA处理的YEplac181的转化效率在rad14::URA3菌株中大大降低。还在具有不同修复能力的单倍体酵母菌株中分析了1,6 - DP加UVA的光生物学效应。结果表明，与亲本野生型菌株相比，核苷酸切除修复途径缺陷的酵母菌株（rad14::URA3）对1,6 - DP加UVA处理高度敏感。证实了1,6 - DP加UVA对野生型酵母的致死作用强烈依赖于氧气，而rad14::URA3突变体的存活仅表现出轻微的氧气依赖性。总之，我们的数据表明，1,6 - DP加UVA诱导的光动力DNA损伤可在原核和真核细胞中通过核苷酸切除修复途径被识别和修复。
