Organic synthesis and recombinant DNA technology were used to situate a putatively premutagenic DNA lesion, O6-methylguanine (O6MeGua), at a specific location in the genomes of two bacterial viruses, M13mp8 and phi X174, and of the bacterial plasmid pBR322. In each genome the first guanine residue in the unique recognition sequence for restriction endonuclease Pst I (5'-C-T-G-C-A-G-3') was replaced with O6MeGua. This was accomplished by ligating a chemically synthesized tetranucleotide, 5'-pTpm6GpCpA-3', into a circular, genome-length heteroduplex in which the four internal nucleotides of the Pst I recognition site had been removed from one strand of the DNA double helix (ligation yield, approximately equal to 50%). It was established that the tetranucleotide was located specifically at the Pst I site and that the presence of O6MeGua rendered the ligation product resistant to cleavage by Pst I. Sensitivity of the genome to Pst I was restored upon treatment with purified Escherichia coli O6MeGua DNA-methyltransferase, a repair protein that removes the methyl group from DNA-bound O6MeGua. This result, in combination with other data, showed unambiguously that O6MeGua was incorporated with high yield into the Pst I recognition sequence.

Sterigmatocystin (ST), a potent hepatocarcinogen, was covalently bound to calf thymus DNA by incubation in the presence of phenobarbital-induced rat liver microsomes. Acid hydrolysis of ST-modified DNA liberated a major guanine-containing adduct, present in DNA at an estimated level of 1 ST residue per 100-150 nucleotides. The adduct was isolated by high-pressure liquid chromatography and subjected to structural analysis. Spectral and chemical data identified the adduct as 1dihydro-2(NI-guanyl)--hydroxysterigmatocystin, the guanine and hydroxyl moieties being in a trans configuration. The structure and stereochemistry of this adduct indicated that the exo-ST-1,2-oxide was the metabolite that reacted with DNA, and the quantitative yield of adduct indicated that this metabolite was a major product of the in vitro metabolism of ST.Sterigmatocystin 'ST) is a carcinogenic mycotoxin produced as a secondary metabolite by Aspergillus, Penicillium, and Bipolaris species (1, 2). ST is acutely toxic to the liver of most animals tested (3, 4), and its carcinogenicity has been demonstrated with organ specificity varying with species and route and frequency of administration (5-9). In rats, ST induces hepatocellular carcinomas after oral administration (6) or intraperitoneal injection (5) and squamous cell carcinomas after repeated application to the skin (7). Despite its potent toxic and carcinogenic properties in animals, the importance of ST as a human health hazard is unknown because surveillance programs have detected its presence in foods only infrequently and at low concentrations even though ST-producing fungi are widely distributed (10).Nonetheless, ST is of interest as a model compound for cancer induction because of its structural similarity to aflatoxin B1 (AFB1). In rats and monkeys, the lethal potency of ST is about 1/10th that of AFB1 (3), and ST is between 1 and 2 orders of magnitude less potent as a hepatocarcinogen for the rat (5, 6). A comparable quantitative difference exists in the toxicities and mutagenicities of ST and AFB1 in Salmonella typhimurium (11,12).In contrast to the large literature on AFB1, little information has been published on the metabolism and biochemical effects of ST. It has been shown that a large portion of the dose administered to monkeys is converted to a ST-glucuronide (13) and that metabolic activation is required for the toxicity and mutagenicity of ST in bacteria and some cultured cells (11,12,(14)(15)(16). Mammalian cells in culture exposed to ST display nucleolar aberrations, inhibited mitosis, inhibited uptake of thymidine and uridine, and stimulated DNA repair synthesis (17)(18)(19)(20). ST also has been demonstrated to inhibit RNA synthesis in rat liver (21) (N7-guanyl)-l-hydroxysterigmatocystin (ST-N7-Gua). § MATERIALS AND METHODSST was metabolically activated in the presence of calf thymus DNA by using phenobarbital-induced rat liver microsomes. The incubation mixture (400 ml; divided into eight 50-ml portions) included approximately 1 mg of microsomal protein...

The synthesis and characterization of the oligomer 5'-dTp(06-Me)GpCpA-3' by the modified triester procedure is described, representing the preparation of a DNA fragment containing a base specifically covalently modified by a carcinogen. With use of the tools of genetic engineering, this tetramer will be substituted for a S'-TpGpCpA-3' portion of the DNA of bacterial virus 4x1 74 in order to study the effect on replication of a well-characterized chemical modification of DNA at an exactly known point. The presence of @-methylguanine in the oligomer is shown to inhibit the enzyme activities of snake venom phosphodiesterase and endonuclease PI.

No abstract

No abstract