Deamination of Cytosine-containing Pyrimidine Photodimers in UV-irradiated DNA

The realization that cytosine in cyclobutyl pyrimidine dimers rapidly deaminates to uracil raised the possibility that this chemical transformation, rather than an enzymatic polymerase error, is the major mutagenic step in UV mutagenesis. We have established a sensitive bioassay system that enabled us to determine the rate of deamination of cytosine in cyclobutyl pyrimidine dimers in plasmid DNA. This was done by in vitro UV irradiation and deamination of a plasmid carrying the cro gene, followed by photoreactivation, and assaying uracils in DNA by their ability to cause Cro− mutations in an indicator strain that was deficient in uracil DNA N-glycosylase. DNA sequence analysis revealed that 27 out of 29 Cro− mutants carried GC → AT transitions, as expected from deamination of cytosine. Deamination of cytosines in the cro gene in UV-irradiated plasmid pOC2 proceeded at 37°C with first-order kinetics, at a rate of (3.9 ± 0.6) × 10−5 s−1, corresponding to a half-life of 5 h. Physiological salt conditions increased the half-life to 12 h, whereas decreasing the pH increased deamination. The temperature dependence of the rate constant yielded an activation energy of 13.6 ± 3.3 kcal/mol. These kinetics data suggest that deamination of cytosine-containing dimers is too slow to play an important role in UV mutagenesis in Escherichia coli. However, it is likely to play an important role in mammalian cells, where the mutagenic process is slower.