Abstract
We have constructed single-stranded, M13-based vectors that contain a specifically located thymine-thymine pyrimidine-pyrimidone(6-4) UV photoproduct and have used these to estimate the frequency and accuracy of DNA replication past this adduct in uvrA6 cells of Escherichia coli. Both the normal and the Dewar valence photoisomer of the (6-4) adduct were studied. In the absence of SOS induction, vectors carrying the photoproducts were rarely replicated; relative to the lesion-free control, 1.9% of vectors carrying the normal (6-4) isomer produced plaques, and with the Dewar valence isomer the proportion was 0.4%. In SOS-induced cells, these frequencies rose to 22.1% and 12.3%, respectively. The error frequency of replication past the normal isomer in SOS-induced cells was high; in a random sample of 185 progeny phage analyzed, 169 (91%) contained mutations, all of which were targeted. Equally striking, a high proportion of the mutations (158/169; 93%) were of only one type, namely 3' T----C transitions. Both the error frequency and the specificity were much reduced with the Dewar valence isomer; overall, 74/140 (53%) of the phage analyzed were mutant, and of these only 34 (46%) entailed the 3' T----C transition. We speculate that the high error frequency and specificity arise from the formation of a stable T-G base pair, involving hydrogen bonds at O-2 and N-3 in the pyrimidone ring. Potential hydrogen bonds at these sites are coplanar in the normal but not in the Dewar isomer, perhaps explaining the reduced specificity of mutagenesis with the latter adduct.