A G to T mutation has been observed at the third position of codon 249 of the p53 tumor-suppressor gene in over 50% of the hepatocellular carcinoma cases associated with high exposure to aflatoxin B1 (AFB1). Hypotheses have been put forth that AFB1, in concert with hepatitis B virus (HBV), may play a role in the formation of, and͞or the selection for, this mutation. The primary DNA adduct of AFB1 is 8,9-dihydro-8-(N 7 -guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua), which is converted naturally to two secondary lesions, an apurinic site and an AFB1-formamidopyrimidine (AFB1-FAPY) adduct. AFB1-FAPY is detected at near maximal levels in rat DNA days to weeks after AFB1 exposure, underscoring its high persistence in vivo. The present study reveals two striking properties of this DNA adduct: (i) AFB1-FAPY was found to cause a G to T mutation frequency in Escherichia coli approximately 6 times higher than that of AFB1-N7-Gua, and (ii) one proposed rotamer of AFB1-FAPY is a block to replication, even when the efficient bypass polymerase MucAB is used by the cell. Taken together, these characteristics make the FAPY adduct the prime candidate for both the genotoxicity of aflatoxin, because mammalian cells also have similar bypass mechanisms for combating DNA damage, and the mutagenicity that ultimately may lead to liver cancer. Aflatoxin B 1 (AFB 1 ), one of the most potent known liver carcinogens, is produced by the common soil fungus Aspergillus flavus. Exposure to this toxin is high in regions of the world where certain foods are improperly stored (1). Hepatitis B virus (HBV) is also common in these regions, and epidemiological evidence indicates that there is a synergistic interaction between AFB 1 exposure and HBV infection on the induction of hepatocellular carcinoma (HCC). In over 50% of HCC cases studied in these areas, a characteristic G to T mutation is observed at the third position of codon 249 of the p53 tumorsuppressor gene (2, 3). Whether this specific sequence is an exceptional target for mutations caused by AFB 1 or whether the mutation is selected for once it occurs remains to be determined. However, each of these scenarios shares the fundamental early step involving generation of a G to T mutation.There is substantial evidence that AFB 1 -induced G to T mutations in cellular ras genes may also be a step in transformation of normal cells to malignant cells (4-6). In humans these mutations occur at the first and second positions of codon 12 in the Ha-ras protooncogene (7) and they are in sequence contexts similar, but not identical, to that of codon 249 in p53.Many studies have defined the mutational spectrum produced after exposure of cells to either the epoxide, which is the toxicologically relevant natural metabolite of AFB 1 (8), or to other electrophilic derivatives that serve as models for the epoxide (9). The G to T mutation is predominantly observed (2,3,(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Studies of mutational landscapes, by their nature, do not elucidate which specific chemical form o...

Base excision repair glycosylases locate and remove damaged bases in DNA with remarkable specificity. The MutY glycosylases, unusual for their excision of undamaged adenines mispaired to the oxidized base 8-oxoguanine (OG), must recognize both bases of the mispair in order to prevent promutagenic activity. Moreover, MutY must effectively find OG:A mismatches within the context of highly abundant and structurally similar T:A base pairs. Very little is known about the factors that initiate MutY’s interaction with the substrate when it first encounters an intrahelical OG:A mispair, or about the order of recognition checkpoints. Here, we used structure–activity relationships (SAR) to investigate the features that influence the in vitro measured parameters of mismatch affinity and adenine base excision efficiency by E. coli MutY. We also evaluated the impacts of the same substrate alterations on MutY-mediated repair in a cellular context. Our results show that MutY relies strongly on the presence of the OG base and recognizes multiple structural features at different stages of recognition and catalysis to ensure that only inappropriately mispaired adenines are excised. Notably, some OG modifications resulted in more dramatic reductions in cellular repair than in the in vitro kinetic parameters, indicating their importance for initial recognition events needed to locate the mismatch within DNA. Indeed, the initial encounter of MutY with its target base pair may rely on specific interactions with the 2-amino group of OG in the major groove, a feature that distinguishes OG:A from T:A base pairs. These results furthermore suggest that inefficient substrate location in human MutY homologue variants may prove predictive for the early onset colorectal cancer phenotype known as MUTYH-Associated Polyposis, or MAP.

Aflatoxin B(1) (AFB(1)), the most potent member of the aflatoxin family of hepatocarcinogens, upon metabolic activation reacts with DNA and forms a population of covalent adducts. The most prevalent adduct, 8,9-dihydro-8-(N(7)-guanyl-)-9-hydroxyaflatoxin (AFB(1)-N(7)-dG), as well as the AFB(1) formamidopyrimidine adduct (AFB(1)-FAPY), resulting from imidazole ring opening of the major adduct, are thought to be responsible for mutations caused by AFB(1). The AFB(1)-N(7)-dG lesions are rapidly removed in Escherichia coli and mammals, whereas the AFB(1)-FAPY lesions persist in mammalian cells, which along with the higher stability of this lesion suggests that AFB(1)-FAPY might significantly contribute to the observed toxicity and carcinogenicity of AFB(1) in higher organisms. Other workers have shown in vitro evidence that AFB(1)-FAPY lesions are substrates for both nucleotide excision repair (NER) and base excision repair (BER). The present study, done in vivo, utilized a modified host cell reactivation assay and showed that AFB(1)-FAPY lesions are preferentially repaired in E.coli by NER. Comparisons of repair in wild-type, NER-deficient (uvrA), BER-deficient (mutM) and NER/BER double mutant E.coli strains transformed with plasmids enriched for AFB(1)-N(7)-dG or AFB(1)-FAPY lesions indicate that both lesions are efficiently repaired by NER-proficient cells (both wild-type and BER-deficient strains). We have found that the level of activity of the reporter gene is significantly affected by the presence of either lesion in NER-deficient strains due to the lack of repair. This effect is similar in NER-deficient and NER/BER-deficient strains indicating that BER (specifically in the strains we investigated) does not contribute significantly to the repair of these lesions in vivo. Consistent with this finding, in vitro analysis of AFB(1)-FAPY adduct excision by purified MutM and its functional analog human 8-oxoguanine DNA glycosylase using site-specifically modified oligonucleotides indicates that this lesion is a poor substrate for both proteins compared with canonical substrates for these enzymes, such as 7,8-dihydro-8-oxoguanine and methylformamidopyrimidine.

Modified oligonucleotides that contain a sulfur atom in place of an oxygen atom have proven enormously valuable in studies of nucleic acid structure and function, 1-3 protein-nucleic acid interactions, 4-6 and antisense gene therapy. 7 Virtually every oxygen atom on the bases, 1,4 the sugar, 2,5,7 and the phosphoryl group 3,6 has been replaced by sulfur. Some of these nucleotide analogues have provided insight into the most intricate details of biological function. 8 Substitution of the 2′-hydroxyl group in RNA by a mercapto (SH) group has received relatively little attention despite the potential use of 2′-deoxy-2′-mercaptonucleosides as probes for the role of the 2′-hydroxyl in RNA structure and function. Although the first 2′-deoxy-2′-mercaptonucleosides were synthesized more than two decades ago, 9 only recently has the synthesis of an RNA dinucleotide containing 2′-deoxy-2′-mercaptouridine been reported. 10 The synthesis employed phosphate triester methodology in which 5′-O-(9-phenylxanth-9-yl)-2′-deoxy-2′-(9-(p-anisyl)xanthen-9-ylthio)uridine-3′-O-(2-chlorophenyl)phosphate was coupled to 2′,3′-di-O-acetyluridine. This approach used relatively strong acidic conditions (0.1 M HCl) to remove the 2′-[9-(p-anisyl)xanthen-9-yl] sulfur protecting group and may therefore be problematic in the synthesis of longer oligonucleotides due to the tendency of adenosine and guanosine to depurinate under acidic conditions. 11 Although phosphate triester methodology is useful for large scale solution synthesis of oligonucleotides, solid phase phosphoramidite methodology is generally the method of choice for most biochemical investigations because it enjoys near quantitative coupling yields and fewer side products. 12 Thus, the development of chemistry for the incorporation of 2′-mercaptonucleosides into RNA and DNA via the phosphoramidite approach would facilitate further investigation of nucleic acid structure and function. Herein, we report the synthesis of appropriately protected phosphoramidites that are suitable for the incorporation of 2′-deoxy-2′-mercaptocytidine and 2′-deoxy-2′-mercaptouridine into oligonucleotides. Methods for the postsynthetic protection and deprotection of the mercapto group via disulfide exchange chemistry are also described.Standard preparation of nucleoside synthons used in solid phase synthesis of oligonucleotides includes protection of the 2′-and 5′-hydroxyls as tert-butyldimethylsilyl and dimethoxytrityl ethers, respectively, protection of the heterocyclic amines as amides, and phosphitylation of the 3′-oxygen to the -cyanoethyl N,N-diisopropylphosphoramidite. 13 Protection of a 2′-mercapto group during solid phase synthesis is necessary to prevent side reactions due to the redox and nucleophilic properties of sulfur. Furthermore, because of the potential intramolecular S N reactions at either the 1′-carbon 14 or 3′-phosphorus 15 under basic conditions, it is important that the sulfur protecting group remain stable during removal of the base labile groups. The most commonly used 2′-hydroxyl p...