In vivo deamination of cytosine-containing cyclobutane pyrimidine dimers in E. coli: a feasible part of UV-mutagenesis

We have estimated in vivo deamination rates for cytosines in cyclobutane pyrimidine dimers (CPD or PyPy) in UV-irradiated E. coli deficient in uracil DNA glycosylase. The protocol consisted of UV-irradiation, holding in buffer to allow for deamination of cytosines in CPDs and photoreversal (PR) to establish uracils where cytosines in CPD deaminated. The deamination rate at TC photoproducts targeting glutamine tRNA suppressor mutations was estimated from the increase in the mutation frequency after PR (MFPR) that developed as UV-irradiated cells were held before PR. Evidence suggested that an earlier study with this protocol under-estimated the deamination rate at sites producing the same mutations in an E. coli B/r strain. With a K12 strain, where the targeting apparently is principally by CPD and not (6-4) photoproducts, a larger rate of k = 0.0091min-1 at 42 deg. C resulted. The dark assay for MF also increased significantly with time for deamination consistent with a model for efficient mutation by translesion synthesis at uracil-containing CPD. In addition, we used a strain constructed by Cupples and Miller in which β-galactosidase was inactive because -GGG- was at codon 461 and would revert to Lac+ only when replaced by -GAG- or -GAA- for glutamate. CC photoproducts at this target site in the opposite DNA strand could reveal effects of first and second deaminations in the same CPD. MFPR for Lac+ mutations increased and then decreased as a function of deamination time (at six temperatures 36-48 deg. C). Fitting an approximate model equation that distinguished two different deamination rates to these data suggested a first deamination producing Lac+ at a rate about eight-fold less than a second deamination restoring the Lac- phenotype. We conclude that deamination, changing a cytosine-containing CPD to a uracil-containing CPD, could be an integral part of UV-induced C-to-T mutations