Implication of uracil in spontaneous mutagenesis on a single-stranded shuttle vector replicated in mammalian cells

Abstract

Almost all spontaneous point mutations found on a single-stranded shuttle vector after its transfection and replication in monkey cells were located at cytosine residues. In order to understand this very specific type of targeting we have studied the possible implication of uracil residues in the induction of these spontaneous mutations. The single-stranded shuttle vector pCF3A carrying the supF tRNA gene as a mutagenesis target has been allowed to replicate in mammalian COS7 cells, mutations being screened in bacteria using the beta-galactosidase assay. Progenies from untreated DNA and DNA treated with the uracil-DNA glycosylase prior to transfection were analyzed to determine the amount and classes of mutations. While spontaneous mutation frequency was 9.7 x 10(-4) for control DNA, single-stranded vector treated with the E. coli uracil-DNA glycosylase exhibited a reduced mutation frequency of about 30%. The abolished mutations were mainly confined to the cytosine to thymine transitions for which a decrease by a factor of 5 was indeed observed. This finding fits well with the fact that it is usually admitted that uracil pairs with adenine, indicating therefore that approximately 30% of spontaneous mutations observed in our experimental conditions and 80% of C to T transitions may be due to the presence of uracil instead of cytosine.