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. 2012 Mar;40(6):2494-505.
doi: 10.1093/nar/gkr1095. Epub 2011 Nov 25.

Lack of recognition by global-genome nucleotide excision repair accounts for the high mutagenicity and persistence of aristolactam-DNA adducts

Victoria S Sidorenko  1 , Jung-Eun Yeo, Radha R Bonala, Francis Johnson, Orlando D Schärer, Arthur P Grollman
Affiliations

Lack of recognition by global-genome nucleotide excision repair accounts for the high mutagenicity and persistence of aristolactam-DNA adducts

Victoria S Sidorenko et al. Nucleic Acids Res. 2012 Mar.

Abstract

Exposure to aristolochic acid (AA), a component of Aristolochia plants used in herbal remedies, is associated with chronic kidney disease and urothelial carcinomas of the upper urinary tract. Following metabolic activation, AA reacts with dA and dG residues in DNA to form aristolactam (AL)-DNA adducts. These mutagenic lesions generate a unique TP53 mutation spectrum, dominated by A:T to T:A transversions with mutations at dA residues located almost exclusively on the non-transcribed strand. We determined the level of AL-dA adducts in human fibroblasts treated with AA to determine if this marked strand bias could be accounted for by selective resistance to global-genome nucleotide excision repair (GG-NER). AL-dA adduct levels were elevated in cells deficient in GG-NER and transcription-coupled NER, but not in XPC cell lines lacking GG-NER only. In vitro, plasmids containing a single AL-dA adduct were resistant to the early recognition and incision steps of NER. Additionally, the NER damage sensor, XPC-RAD23B, failed to specifically bind to AL-DNA adducts. However, placing AL-dA in mismatched sequences promotes XPC-RAD23B binding and renders this adduct susceptible to NER, suggesting that specific structural features of this adduct prevent processing by NER. We conclude that AL-dA adducts are not recognized by GG-NER, explaining their high mutagenicity and persistence in target tissues.

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Figures

Figure 1.
Figure 1.
Structures of AA and dA-AL and dG-AL adducts. The DNA adducts are formed by cellular reduction of the nitro group, hydroxy lactone formation and activation to react with the exocyclic amine groups of dA and dG.
Figure 2.
Figure 2.
AAII cytotoxicity study. (A–F) XP-, CS-deficient and control normal human fibroblasts cell lines were treated with 5–200 µM AAII for 72 h with the following ATP level measurements. Filled circles indicate the control normal human fibroblasts cell line, open circles—the deficient cell lines corresponding to the panel names, filled triangles—complemented cell lines if available. In the case of CSB-deficient cell lines, the complementation of the deficient cell line was achieved through pc3.1 plasmid vector harboring the cDNA of wild-type or ATPase mutant ERCC6 gene. On the F, filled squaresindicate the CSB-deficient cell line complemented with ATPase mutant gene. To correct the abnormal phenotype XPA-deficient cells are transfected with the full-length cDNA of the XPAC gene. The XPD cell line is transfected with p2E-ER2, a complementary DNA expression construct of the ERCC2 (XPD) gene, to correct the abnormal phenotype. The point for each AAII dose corresponds to the ATP ratio between treated and untreated cells and presents mean ± standard deviation for at least six measurements in two independent experiments.
Figure 3.
Figure 3.
AAII genotoxicity study. XP-, CS-deficient, normal fibroblast and complemented cell lines if available were treated with 20, 50, 100 µM AAII for 48 h. DNA was isolated and analyzed for adduct levels with 32P-post-labeling assay. (A–F) – The dependence of dG- and dA-ALII adduct levels from the treatment AAII dose. Control indicates the control human fibroblasts cell line. XPA-, XPD-, XPG-, CSA-, CSB-, CSBmut-, XPC-deficient human fibroblasts cell lines. XPA+, XPD+, CSB+ are corresponding complemented cells. All treatments for every cell line were done in triplicate and the results are presented as means ± standard deviations. (G) The fragment of a 30% polyacrylamide gel after 32P-labeling of DNA adduct nucleosides. 1–3, XPA-deficient cell line treated with 20, 50, 100 µM AAII for 48 h; 4–6, XPA complemented cell line treated the same way; st, The standard mixture of 24-mer oligonucleotides containing 15 fmol of each of the single dG-ALII or dA-ALII, the upper and lower bands, respectively. Each standard band corresponds to 1 adduct/106 nucleotides for 5 µg DNA. For each DNA digestion, at least three standard mixtures were used.
Figure 4.
Figure 4.
NER dual incision activity on dG-AAF, dG-AF and dA-ALII containing plasmids. Plasmids containing site-specific dG-AAF (lane 1), dG-AF (lane 2) or dA-ALII (lanes 3–6) residues were incubated with a HeLa cell extract. Excision products containing were detected by annealing to complementary oligonucleotides with a 4G overhang, which served as a template for end-labeling with [α-32P] dCTP and sequenase. The reaction products were resolved on a 14% denaturing polyacrylamide gel. A low molecular weight ladder from NER was used as a size marker and the position of 25 and 34 nt bands are indicated. Asterisks denotes non-specific, NER-independent bands.
Figure 5.
Figure 5.
Determination of binding affinities of XPC-RAD23B binding to dG-ALII and dG-AAF containing oligonucleotides. (A) Lesion-containing 44-mer oligonucleotides (4 nM) were annealed to a complement containing a 5′ Cy5 fluorescent label and incubated with different concentrations (0–125 nM) of XPC-RAD23B for 30 min. The reactions were analyzed on a 5% native polyacrylamide gel and visualized on a Typhoon imaging system. Two representative gels using C209-ALII and C209-ALII with mismatch are shown. (B) Determination of Kd values: the percentage of bound (DNA+XPC-RAD23B) and non-bound fractions (DNA) were determined using Image Quant TL. Kd were calculated from at least three independent experiments and the data analyzed using sigma plot. Standard deviations are indicated by error bars.
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