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

Abstract

We have estimated in vivo deamination rates for cytosines in cyclobutane pyrimidine dimers (CPD or Py ▪Py) 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 T ▪C photoproducts targeting glutamine tRNA suppressor mutations was estimated from the increase in the mutation frequency after PR (MF PR) 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 = 0.0091 min −1 at 42 °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. C ▪C photoproducts at this target site in the opposite DNA strand could reveal effects of first and second deaminations in the same CPD. MF PR for Lac + mutations increased and then decreased as a function of deamination time (at six temperatures 36–48 °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.