Endemic (Balkan) nephropathy (EN),environmental mutagen ͉ p53 mutation ͉ urothelial cancer ͉ DNA adduct

Oxidative DNA damage is generated by a variety of environmental and endogenous agents, including ionizing radiation, certain chemicals, and products of aerobic metabolism (1). 8-oxoG 1 is one of the most abundant forms of oxidative DNA damage (2). Due to its ability to form a Hoogstein-type base pair with adenine (3), 8-oxoG is miscoding (4) and mutagenic, resulting in G3 T transversions in bacterial and eukaryotic cells (5, 6). The potential harmful effects of this lesion are avoided by base excision repair. In Escherichia coli, formamidopyrimidine-DNA glycosylase (Fpg, EC 3.2.2.23) removes 8-oxoG, Me-FaPy, and several structurally related lesions from damaged DNA (7,8). Fpg is a component of the "GO system" that includes MutY, a mismatch adenine-DNA glycosylase, and MutT, an 8-oxodGTPase (9, 10); E. coli strains deficient in any of these genes are strong mutators (11).Fpg shares significant sequence homology with endonuclease VIII (Nei) of E. coli (12). Both proteins belong to a family unrelated by sequence or tertiary structure to a larger family of DNA glycosylases, for which the prototype is endonuclease III (Nth) (13,14). The substrate specificity of Fpg differs significantly from Nei (7, 8, 15) but closely resembles that of the eukaryotic 8-oxoguanine-DNA glycosylase, Ogg1, a member of the Nth family (14,16,17). Fpg also possesses AP lyase activity, nicking the phosphodiester backbone of DNA at the site of the lesion. Base excision by Fpg is followed immediately by two ␤-elimination steps, resulting in a single nucleotide gap flanked by phosphate termini (7). A Schiff base intermediate, involving Pro-1 of the enzyme and C1Ј of the damaged nucleotide, forms early in the reaction sequence and can be reductively trapped by treatment with NaBH 4 forming a stable covalent complex (18,19). The mechanism of cleavage is similar to that of Nei (15,20), but not to that of Ogg1 where only one ␤-elimination occurs, and the efficiency of the elimination step is very low compared with base excision (16,17).Comparing the structures of Fpg, Nei, and Ogg1 provides a unique opportunity to analyze features of damage recognition and catalysis common to DNA glycosylases/AP lyases. The presence of DNA enhances the analytic power of the model by revealing the precise nature of enzyme-DNA interactions. The structure of the human Ogg1 catalytic domain complexed to DNA has been solved (21, 22), as has the structure of E. coli Nei covalently cross-linked to DNA by NaBH 4 (23). The structure of Fpg from Thermus thermophilus HB8 (Tth-Fpg) has recently been solved in the absence of DNA (24). Although mechanisms for lesion recognition and catalysis by Fpg have been suggested on the basis of this structure and on earlier biochemical studies of E. coli Fpg (8,18,24,25), many questions remain unanswered regarding the mode of Fpg-DNA interactions and the catalytic reaction mechanism of this important DNA repair protein.To investigate the mechanisms of Fpg-DNA interactions, we have utilized NaBH 4 reduction of the Schiff base intermediate t...

contributed equally to this work Endonuclease VIII (Nei) of Escherichia coli is a DNA repair enzyme that excises oxidized pyrimidines from DNA. Nei shares with formamidopyrimidine-DNA glycosylase (Fpg) sequence homology and a similar mechanism of action: the latter involves removal of the damaged base followed by two sequential b-elimination steps. However, Nei differs signi®cantly from Fpg in substrate speci®city. We determined the structure of Nei covalently crosslinked to a 13mer oligodeoxynucleotide duplex at 1.25 A Ê resolution. The crosslink is derived from a Schiff base intermediate that precedes b-elimination and is stabilized by reduction with NaBH 4 . Nei consists of two domains connected by a hinge region, creating a DNA binding cleft between domains. DNA in the complex is sharply kinked, the deoxyribitol moiety is bound covalently to Pro1 and everted from the duplex into the active site. Amino acids involved in substrate binding and catalysis are identi®ed. Molecular modeling and analysis of amino acid conservation suggest a site for recognition of the damaged base. Based on structural features of the complex and site-directed mutagenesis studies, we propose a catalytic mechanism for Nei.

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This study was designed to establish the TP53 mutational spectrum of aristolochic acid (AA), examined in the context of endemic (Balkan) nephropathy, an environmental disease associated with transitional cell (urothelial) carcinomas of the upper urinary tract (UUC). Tumor tissue was obtained from residents of regions in Bosnia, Croatia and Serbia where endemic nephropathy has been prevalent for over 50 years. Fifty-nine TP53 mutations were detected in 42 of the 97 tumors analyzed. Mutational spectra were dominated by A:T to T:A transversions with the mutated adenines located almost exclusively on the nontranscribed strand. This marked strand bias is attributed to selective processing of aristolactam-dA adducts by transcription-coupled nucleotide excision repair. Hotspots for A:T to T:A mutations include codons 131 and 179 and the 5 0 -AG acceptor splice site of intron 6. The unique TP53 mutational signature for AA identified in this study can be used to explore the hypothesis that botanical products containing this human carcinogen and nephrotoxin are responsible, in part, for the high prevalence of UUC and chronic renal disease in countries where Aristolochia herbal remedies traditionally have been used for medicinal purposes.