In vivo evidence that UV-induced C-->T mutations at dipyrimidine sites could result from the replicative bypass of cis-syn cyclobutane dimers or their deamination products.
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.
Jiang
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Department of Chemistry, Washington University, St. Louis, Missouri 63130.
Taylor
J S
JS
D000586:Amination; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D003553:Cystine / Q000235:genetics* / Q000378:metabolism; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D003641:Deamination; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010777:Photochemistry; D011740:Pyrimidine Dimers / Q000235:genetics*; D013014:SOS Response, Genetics; D013936:Thymidine / Q000235:genetics* / Q000378:metabolism; D014466:Ultraviolet Rays
D004269:DNA, Bacterial; D011740:Pyrimidine Dimers; D003553:Cystine; D013936:Thymidine