In Vivo Evidence That UV-Induced C → T Mutations at Dipyrimidine Sites Could Result from the Replicative Bypass of Cis-Syn Cyclobutane Dimers or Their Deamination Products

The major mutations induced by UV light are C ₒ T transitions at dipyrimidines and arise from the incorporation of A opposite the C of dipyrimidine photoproducts. The incorporation of A has most often been explained by the known preference of a polymerase to do so opposite noninstructional DNA damage such as an abasic site (A rule). There are also mechanisms that suppose, however, that cis-syn dipyrimidine photodimers are instructional. In one such mechanism (tautomer bypass), the incorporation of A is directed by the tautomer of a C of a dimer that is equivalent in base-pairing properties to U [Person et al. (1974) Genetics 78, 1035–1049]. In another mechanism (deamination bypass), the incorporation of A is directed by a U of a dimer that results from the deamination of the C of a dimer [Taylor & O'Day (1990) Biochemistry 29, 1624–1632]. The viability of these mechanisms was tested by obtaining the mutation spectrum of a TU dimer in Escherichia coli by application of a standard method for site-directed mutagenesis. To this end, a 41-mer containing a site-specific TU dimer was constructed via ligation of a dimer-containing decamer that was produced by triplet-sensitized irradiation and used to prime DNA synthesis on a uracil-containing (+) strand of an M13 clone containing a double mismatch opposite the dimer. The reaction mixture was used to transfect a uracil glycosylase proficient, photoproduct repair deficient E. coli host, and all progeny phage weakly hybridizing to the parental (+) or (−) strands were sequenced. Under non-SOS conditions the TU dimer almost completely blocked replication, while under SOS conditions it directed the incorporation of two As with much higher specificity (96%) than would an abasic site. The implications of these results to the mechanism of the UV-induced TC → TT mutation, and by extension to the CT → TT, CC → TC., CC → CT, and the tandem CC → TT mutations, are discussed.