MUTAGENIC CONSEQUENCES OF DEFINED LESIONS IN DNA

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The mutagenic consequences of introducing defined lesions into DNA are being examined in a mutational system capable of detecting a wide spectrum of mutational events, both at and some distance away from the actual site of damage. The system has first been applied to depurination, the loss of a purine base from DNA. This is a frequent spontaneous lesion as well as a common intermediate in the repair of many other types of DNA damage. Depurination is highly mutagenic, as determined by transfecting depurinated MI3mp2 DNA into SOS-induced competent E. coli cells. DNA sequence analysis of 211 mutants demonstrates that most mutations are base substitutions reflecting insertion of dAMP opposite the non-coding abasic site and resulting in characteristic transversions. Such transversions are frequently observed in vivo for several damaging agents which produce bulky DNA adducts, suggesting that such adducts may produce mutations either through an abasic site intermediate, or by simply precluding template base hydrogen bonding. The second defined lesion is a uracil residue in DNA, resulting from the heat-induced deamination of cytosine. This is suggested to be the second most frequent spontaneous lesion in DNA, representing a significant challenge to a cell at 37 degrees C. For these studies, the first in vitro assay for reversion of a proline codon (CCC) has been developed. The assay is both highly specific and very sensitive. Using this and the forward-mutation assay, the rate constant of C deamination has been determined at physiological pH and salt concentrations and 3 37 degrees C. The sequence specificity of C deamination has just come under examination. Eventually, the contribution of cross-strand protonation by various adducts in double-stranded DNA, leading to C deamination, will be investigated.