Frequency and Spectrum of Mutations Produced by a Single Cis-Syn Thymine-Thymine Cyclobutane Dimer in a Single-Stranded Vector

We have constructed a single-stranded vector that contains a uniquely located cis-syn T-T cyclobutane dimer by ligating a synthetic oligomer containing this UV photoproduct into M13mp7 viral DNA linearized with EcoRI. In the absence of SOS induction, transfection of a uvrA6 mutant of Escherichia coli with this vector gave very few progeny plaques, and the data imply that a single dimer blocks replication in at least 99.5% of the molecules. In vitro photoreactivation completely abolished this inhibition. Transfection of cells irradiated with UV at 4 J· m-2 to induce the SOS response gave 27% of the number of plaques found with a dimer-free control. Nucleotide sequence analysis of 529 progeny phage showed that translesion synthesis was usually accurate: the normal sequence was found in 93% of them. Where mutations occurred, all were targeted single-nucleotide substitutions, with ≈ 90% being targeted at the 3′ nucleotide of the lesion: of a total of 26 mutations, 15 were 3′ T → A, 8 were 3′ T → C, and 3 were 5′ T → C. No T → G mutations were found. In addition to these results with the normal construct, data were also obtained from vectors in which the M13mp7 cloning site, which forms a hairpin in single-stranded DNA, was present 4 nucleotides on the 3′ side of T-T dimer. These hairpin-containing vectors gave a very similar mutation frequency (8% versus 7%) but altered mutation spectrum: all 12 mutations detected were 3′ T → A transversions, a difference from the previous set of data that is significant (P = 0.03).