Accuracy of replication past the T-C (6-4) adduct

Abstract

The thymine-cytosine pyrimidine-pyrimidone (6-4) adduct has variously been predicted to be among the most and among the least mutagenic of the ultraviolet light photoproducts. We have therefore investigated the frequency and accuracy of DNA replication past this lesion, using a single-stranded M13mp7-based vector with a uniquely located example of this lesion transfected into SOS-induced and uninduced cells of a uvr A6 strain of Escherichia coli. Both the UVC T-C (6-4) adduct and its Dewar valence (UVB) photoisomer were studied. Random samples from non-selective collections of progeny phage were sequenced to determine the nature of the replication events that occurred at or near the site of template damage under SOS conditions. The UVC (6-4) adduct was found to be much less mutagenic than its T-T counterpart, but still much more mutagenic than a cyclobutane dimer; 34% (71 out of 206) of all bypass events yielded mutations, of which all were targeted and 80% (57 out of 71) were 3' C-->T transitions. The Dewar valence photoisomer exhibited reduced specificity and enhanced mutagenicity; 79% (183 out of 233) of the phage progeny were mutants, of which all but one were targeted and 45% (83 out of 183) were 3' C-->T transitions. For the most part, these results are consistent with a model postulating base-pairing between the pyrimidinone (of either the C or T variety) and guanine, via hydrogen bonds at N-3 and O-2 in the UVC, but not the Dewar, isomer. The occurrence of the 3' C-->T transitions, not predicted by this model, shows however that the absence of a methyl group at C-5 also has a significant influence on mutation induction. Both isomers were efficient blocks to replication; less than 1% of these vectors could be replicated in uninduced cells. Following SOS induction the frequency of bypass increased to 24.5% and 12.5% for the UVC and the Dewar isomers, respectively.