We have constructed single-stranded, M13-based vectors that contain a specifically located thyminethymine 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 TOG base pair, involving hydrogen bonds at 0-2 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.Irradiation of DNA with germicidal UV light produces a variety of photoproducts, the chief of which are the cyclobutyl dipyrimidines (dimers) and the pyrimidine-pyrimidone(6-4) adducts (1, 2). We were interested in studying the thymine-thymine (6-4) adduct [5-hydroxy-6-4'-(5'-methylpyrimidin-2'-one)-thymine] as an example of the latter type of photoproduct, for two reasons. (i) We anticipated that it might provide insights into the mechanism determining base selection during translesion synthesis-that is, during chain elongation past the site of a template lesion-and, in general, help in understanding the relation between the structure of a lesion and its mutagenic potential. Previous experiments with cis-syn (3, 4) and trans-syn (5) cyclobutane dimers, and also with abasic sites (6), each located at a T-T site in the same sequence context, suggested that the UV photoproducts often form correct A'T base pairs during translesion synthesis, and that when mutations occur, they might result from base mispairing. We were therefore interested to examine other lesions for evidence of such correct or aberrant base pairing.(ii) We wished to study the T-T (6-4) adduct because ofconflicting conclusions concerning the relative proportions of mutations induced by dimers and adducts in Escherichia coli. Although adducts are collectively 5-to 10-fold less abundant than dimers in UV-irradiated DNA, some evidence (7-9, 25) suggests that they are responsible for the majorit...

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We have investigated the mutagenic properties of an abasic site in DNA by transfecting SOS-induced and uninduced cells of E. coli with a single-stranded M13mp7-based vector that carries a single example of this lesion at one or other of two unique and adjacent sites. Random samples of progeny phage were sequenced to determine the nature of the replication events that occurred at and around these locations. 5% to 7% of the vectors could be replicated in SOS-induced cells, but only 0.1% to 0.7% of them gave plaques in the absence of SOS induction. In SOS-induced cells, 93% and 96% of the phage replicated resulted from the insertion of a nucleotide opposite the abasic site, while the remainder resulted from a targeted omission of a single nucleotide. At one of the sites, nucleotide insertions were 54% dAMP, 25% dTMP, 20% dGMP and 1% dCMP. At the other site they were 80% dAMP, 4% dTMP, 15% dGMP and 1% dCMP. The sequence variation in all but two of the 204 sequences analyzed was restricted to the abasic site itself. In the remaining two, a change at the abasic site was accompanied by a mutation at an immediately flanking nucleotide.

We have transfected SOS-induced and uninduced cells of a uvrA6 strain of Escherichia coli with single-stranded M13mp7-based vectors that carried a single trans-syn T-T cyclobutane dimer at a unique site. Unlike constructs carrying the cis-syn isomer of this lesion, these vectors could be replicated with modest efficiency (14%) in the absence of SOS induction and therefore provided an opportunity to measure directly the influence of such induction on error rate and mutation spectrum. We found that translesion synthesis in the absence of SOS induction was remarkably accurate; only 4% of the replicated bacteriophage contained mutations, which were exclusively targeted single T deletions. In SOS-induced cells, error frequency increased to 11% and the resulting mutations included targeted substitutions and near-targeted single base additions, as well as the T deletions. Replication efficiency was 29% in these conditions. SOS induction therefore leads not only to an enhanced capacity to replicate damaged DNA but also to a marked change in mutation frequency and spectrum.Derepression of the SOS regulon in Escherichia coli results in the production of an altered replication process that copies mutagen-damaged templates with increased efficiency, although with reduced accuracy (13). Such infidelity must, at least in part, be the consequence of the distorted template itself, but it may also reflect inhibition of polymerase functions that promote replication accuracy. It has been argued that inhibition of the 3'-to-5' editing exonuclease, encoded by dnaQ, is a necessary prerequisite for translesion synthesis (12), and data indicating that the RecA protein can indeed inhibit the activity (4, 8) support this argument. Similarly, editing activity does not appear to be involved in induced mutagenesis in normal strains (6,14). It is not known, however, whether the DnaQ gene product is normally part of the altered complex that replicates damaged DNA.A direct measurement of the relative accuracy of translesion synthesis in SOS-induced and uninduced cells has not previously been possible, both because very little bypass is thought to take place without derepression of the SOS regulon and because conventional experiments with mutagen-treated DNA are not well suited for this purpose. An opportunity to make such a direct comparison arose, however, in transfection experiments with a single-stranded M13 hybrid bacteriophage vector that contained a uniquely located trans-syn T-T cyclobutane dimer. Unlike molecules carrying the cis-syn isomer of this lesion, almost none of which are replicated in uninduced cells (1), about 14% of the vector molecules carrying the trans-syn lesion were replicated in the absence of induction, thus allowing the accuracy of translesion synthesis to be determined. A similar determination can, of course, be made for this vector introduced into SOS-induced cells, in which replication efficiency in-* Corresponding author. creased to 29%. At the same time, experiments with such vectors are well suited to the purp...

The mutagenic properties of UV-induced photoproducts, both the cis-syn thymine-thymine dimer (TT) and the thymine-thymine pyrimidine pyrimidone (6-4) photoproduct [T(6-4)T] were studied in mammalian cells using shuttle vectors. A shuttle vector able to replicate in both mammalian cells and bacteria was produced in its single-stranded DNA form. A unique photoproduct was inserted at a single restriction site and after recircularization of the single-stranded DNA vector, this latter was transfected into simian COS7 cells. After DNA replication the vector was extracted from cells and used to transform bacteria. Amplified DNA was finally analyzed without any selective screening, DNA from randomly picked bacterial colonies being directly sequenced. Our results show clearly that both lesions are mutagenic, but at different levels. Mutation frequencies of 2 and 60% respectively were observed with the TT dimer and the T(6-4)T. With the TT dimer the mutations were targeted on the 3'-T. With the T(6-4)T a large variety of mutations were observed. A majority of G-->T transversions were semi-targeted to the base before the 5'-T of the photoproduct. These kinds of mutations were not observed when the same plasmid was transfected directly into SOS-induced JM105 bacteria or when the T(6-4)T oligonucleotide inserted in a different plasmid was replicated in SOS-induced SMH10 Escherichia coil bacteria. These semi-targeted mutations are therefore the specific result of bypass of the T(6-4)T lesion in COS7 cells by one of the eukaryotic DNA polymerases.