Sites of preferential induction of cyclobutane pyrimidine dimers in the nontranscribed strand of lacI correspond with sites of UV-induced mutation in Escherichia coli.

An approach utilizing fluorescence-activated DNA sequencing technology was used to study the position and frequency of UV-induced lesions in the lacI gene of Escherichia coli. The spectrum of sites of UV damage in the NC+ region of the gene was compared with a published spectrum of UV-induced mutation in lacI (Schaaper, R.M., Dunn, R.L., and Glickman, B.W. (1987) J. Mol. Biol. 198, 187-202). On average, the frequency of UV-induced lesions in the nontranscribed strand was higher than that in the transcribed strand in the region analyzed. A large fraction of mutations occurs at sites of UV-induced lesions in the nontranscribed strand, but not in the transcribed strand. This bias is reduced in an excision repair deficient (UvrB-) strain. In addition, mutations occur overwhelmingly at sites where a dipyrimidine sequence is present in the nontranscribed strand. This bias is also markedly reduced in the UvrB- strain. In light of recent work Mellon and Hanawalt (Mellon, I., and Hanawalt, P.C. (1989) Nature 342, 95-98) describing the preferential removal of cyclobutane dimers from the transcribed strand of the expressed lacZ gene in E. coli, our data suggest that preferential strand repair may have a significant effect on mutagenesis.

Colony hybridisation in Escherichia coli: a rapid procedure for determining the distribution of specific classes of mutations among a number of preselected sites.

Colony probe oligonucleotide hybridisation was used for the unambiguous identification of DNA alterations and the determination of distributions and frequencies of forward mutation at the molecular level. To demonstrate the reliability and versatility of this technique, distributions of spontaneous and ethyl methanesulfonate (EMS)-induced mutations have been reproduced using a battery of oligonucleotide probes complementary to specific sites and classes of mutation. These studies are indicative of the diagnostic potential of oligonucleotide colony hybridisation to the characterisation of mutation; oligonucleotide hybridisation used in conjunction with a well studied mutational target provides a rapid and reliable alternative to DNA sequencing for the characterisation of all classes of mutations.