In vivo evidence that UV-induced C .fwdarw. T mutations at dipyrimidine sites could result from the replicative bypass of cis-syn cyclobutane dimers or their deamination products

Abstract

The major mutations induced by UV light are C-->T transitions at dipyrimidines and arise from the incorporation of A opposite the C of dipyrimidine photoproducts. The incorporation of A has most often been explained by the known preference of a polymerase to do so opposite noninstructional DNA damage such as an abasic site (A rule). There are also mechanisms that suppose, however, that cis-syn dipyrimidine photodimers are instructional. In one such mechanism (tautomer bypass), the incorporation of A is directed by the tautomer of a C of a dimer that is equivalent in base-pairing properties to U [Person et al. (1974) Genetics 78, 1035-1049]. In another mechanism (deamination bypass), the incorporation of A is directed by a U of a dimer that results from the deamination of the C of a dimer [Taylor & O'Day (1990) Biochemistry 29, 1624-1632]. The viability of these mechanisms was tested by obtaining the mutation spectrum of a TU dimer in Escherichia coli by application of a standard method for site-directed mutagenesis. To this end, a 41-mer containing a site-specific TU dimer was constructed via ligation of a dimer-containing decamer that was produced by triplet-sensitized irradiation and used to prime DNA synthesis on a uracil-containing (+) strand of an M13 clone containing a double mismatch opposite the dimer. The reaction mixture was used to transfect a uracil glycosylase proficient, photoproduct repair deficient E. coli host, and all progeny phage weakly hybridizing to the parental (+) or (-) strands were sequenced. Under non-SOS conditions the TU dimer almost completely blocked replication, while under SOS conditions it directed the incorporation of two As with much higher specificity (96%) than would an abasic site. The implications of these results to the mechanism of the UV-induced TC-->TT mutation, and by extension to the CT-->TT, CC-->TC, CC-->CT, and the tandem CC-->TT mutations, are discussed.

The Dewar photoproduct of thymidylyl(3′→5′)- thymidine (Dewar product) exhibits mutagenic behavior in accordance with the “A rule”

“…This finding indicates that the Dewar product does not exhibit a preference for insertion of a G opposite its 3Ј T site during TR. It has been reported that during TR in E. coli, an A residue is incorporated opposite abasic sites with a frequency of 77% (18). This is in accordance with the A rule, where an A residue is incorporated opposite an abasic lesion with a 10-fold higher efficiency than a G residue (18).…”

“…It has been reported that during TR in E. coli, an A residue is incorporated opposite abasic sites with a frequency of 77% (18). This is in accordance with the A rule, where an A residue is incorporated opposite an abasic lesion with a 10-fold higher efficiency than a G residue (18). A physical basis for the A rule has been provided by NMR studies showing that A residues opposite abasic sites stack better in the intrahelical configuration than do other bases and cause no helical distortion (22)(23)(24).…”

“…Opposite the abasic site (the prototypical noninstructional DNA lesion), an A residue is incorporated with a frequency of 77% during TR in E. coli (18). This preferential incorporation of an A residue opposite an abasic lesion is referred to as the ''A rule'' (19)(20)(21).…”

Deamination of 5-Methylcytosines within Cyclobutane Pyrimidine Dimers Is an Important Component of UVB Mutagenesis

“…Deamination of C in T-C or C-C dimers leads to formation of T-U or U-U dimers, respectively. Adenines are incorporated with high specificity during bypass of site-specific T-T, T-U, or U-U dimers in vivo (41,45). After deamination of cytosine or 5-methylcytosine within CPDs, DNA polymerase is probably bypassing these CPDs in an error-free manner (20,21,46).…”

Analysis of Damage Tolerance Pathways in Saccharomyces cerevisiae: a Requirement for Rev3 DNA Polymerase in Translesion Synthesis

“…However, an alternative explanation relevant to the plasmid situation that could equally account for the high proportion of 3Gϩ3 pure clones might be the preferential replication and concomitant amplification of the undamaged strand. Lesion-induced strand loss was proposed to explain the prevalence of the undamaged strand in transformants arising from the replication of vectors containing either AAF adducts (20,45) or a single cis-syn cyclobutane thymine dimer (15) in E. coli. It is not possible, with the present constructs, to distinguish between true DA events and lesioninduced strand loss.…”