Most organisms contain several members of a recently discovered class of DNA polymerases (umuC/dinB superfamily) potentially involved in replication of damaged DNA. In Escherichia coli, only Pol V (umuDC) was known to be essential for base substitution mutagenesis induced by UV light or abasic sites. Here we show that, depending upon the nature of the DNA damage and its sequence context, the two additional SOS-inducible DNA polymerases, Pol II (polB) and Pol IV (dinB), are also involved in error-free and mutagenic translesion synthesis (TLS). For example, bypass of N:-2-acetylaminofluorene (AAF) guanine adducts located within the NAR:I mutation hot spot requires Pol II for -2 frameshifts but Pol V for error-free TLS. On the other hand, error-free and -1 frameshift TLS at a benzo(a)pyrene adduct requires both Pol IV and Pol V. Therefore, in response to the vast diversity of existing DNA damage, the cell uses a pool of 'translesional' DNA polymerases in order to bypass the various DNA lesions.

We have constructed pads pS3G-1 and pSG4 that contain single acetylaminofluorene adducts within contiguous runs of three (5'-CCCG'G2G3-3') and four (5'-CG'GGG4T-3') guanine residues, respectively. In Eseherichia coli, the frequency of induced -1 frameshift mutations was strongly dependent on the position of modification: pS3G-G3 was "100-fold and 10-fold more mutagenic than pS3G-GI and pS3G-G2, respectively; pSG4-G4 was "'600-fold more mutagenic than pSG4-Gl. Mutagenesis was SOS-dependent and was markedly reduced in bacteria that were proficient in nucleotide excision repair as compared to a repair-deficient uvrA6 mutant. DNA sequencing showed that -1 fameshift events in pS3G-1 consisted of either targeted mutations (>90% of induced mutations) within the sequence or semitargeted mutations (<10%) in the 5' flanking repetitive cytosine sequence.Semitargeted events, which were observed when acetylamin fluorene modification was at G' and G2, show that a lesion can reduce the fidelity ofreplication at positions 5' to its iocation on the template strand. No semitargeted frameshifts were observed in plasmid pSG4, which lacks a repetitive sequence 5' to the adduct. AAF-induced -1 frameshift mutations, which are recAand umuC/D-dependent, arise through a mechanism different from that responsible for -2 mutations. A classical model for frameshifting within homopolymeric runs (7, 8) wherein strand slippage during DNA synthesis stabilizes extrahelical intermediates is consistent with the sequence specificity of AAF-induced -1 frameshifts. Although the concept of strand slippage within contiguous sequences has endured for many years, the mechanism through which bulky lesions such as the N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene [dG-C8-AAF] adduct might promote this phenomenon remains poorly understood. It has been suggested (9-11) that the observed specificity of -1 frameshifts induced by bulky lesions might be accommodated in a model in which strand slippage occurs after the accurate incorporation of cytosine opposite the lesion; the presence ofguanine residues 5' to the lesion might stabilize slipped intermediates and the normal G-C base-pair terminus of the misaligned intermediate could be extended to produce a mutation. This hypothesis is supported by evidence suggesting that, during in vitro DNA synthesis on unmodified substrates, the frequency of frimeshift mutations increases when a misincorporated base is complementary to the 5' base in the template strand and hence allows the formation of intermediates containing a base-paired terminus (11). The model outlined above makes the prediction that, within a contiguous guanine sequence, frameshift mutagenesis would be specifically enhanced by dG-C8-AAF lesions that contain a guanine residue 5' to the adduct; conversely, AAF lesions at the 5' terminus of contiguous sequences should be nonmutagenic since slippage of a primer bearing a terminal cytosine onto a nonguanine residue 5' to the lesion would form an unstable mismatch.To test these predictions we have co...

Exposure of Escherichia coli to UV light increases expression of NrdAB, the major ribonucleotide reductase leading to a moderate increase in dNTP levels. The role of elevated dNTP levels during translesion synthesis (TLS) across specific replication-blocking lesions was investigated. Here we show that although the specialized DNA polymerase PolV is necessary for replication across UVlesions, such as cyclobutane pyrimidine dimers or pyrimidine(6-4) pyrimidone photoproduct, Pol V per se is not sufficient. Indeed, efficient TLS additionally requires elevated dNTP levels. Similarly, for the bypass of an N-2-acetylaminofluorene-guanine adduct that requires Pol II instead of PolV, efficient TLS is only observed under conditions of high dNTP levels. We suggest that increased dNTP levels transiently modify the activity balance of Pol III (i.e., increasing the polymerase and reducing the proofreading functions). Indeed, we show that the stimulation of TLS by elevated dNTP levels can be mimicked by genetic inactivation of the proofreading function (mutD5 allele). We also show that spontaneous mutagenesis increases proportionally to dNTP pool levels, thus defining a unique spontaneous mutator phenotype. The so-called "dNTP mutator" phenotype does not depend upon any of the specialized DNA polymerases, and is thus likely to reflect an increase in Pol III's own replication errors because of the modified activity balance of Pol III. As up-regulation of the dNTP pool size represents a common physiological response to DNA damage, the present model is likely to represent a general and unique paradigm for TLS pathways in many organisms.