Mutation Research, 269 (1992) 285 -299 285 © 1992 Elsevier Science Publishers B.V. All rights reserved 0027-5107/92/$05.00 MUT 05157 The distribution of UV damage in the lad gene of Escherichia coli: Correlation with mutation spectrum Evelyne Sage, Elisabeth Cramb and Barry W. Glickman * York Unicersity, Department of Biology, North York, Ont. M3J IP3, Canada (Revision received 9 April 1992) (Accepted 10 April 1992) Keywords: Cyclobutane pyrimidine dimer; (6-4) Photoproducr TI DNA pol,~er~se; T4 DNA polymerase 3'-5' exonuclease; Laci gene; Automated DNA sequencer; DNA polymerase Summary We have determined the UV (254 nm) damage distribution in the transcribed and non-transcribed strands of the i-d region of the Escherichia coli lad gene. The locations of replication blocking lesions were revealed as termination sites of T7 DNA polymerase and/or T4 DNA polymerase 3'-5' exonucle- ase. Termination products, i.e. both cyclobutane pyrimidine dimers and 6-4 photoproducts, were resolved and analysed on an automated DNA sequencer. These two major photoproducts are not randomly distributed along the gene, but occur in clusters, in longer runs of pyrimidines. We also have compared the UV damage distribution with the previously reported UV-induced base substitutions in the same region. Mutational hotspots, in both repair-deficient and repair-proficient strains of E. coil, are all located in stretches of pyrimidines~ and thus correlate well with the distribution of photolesions. One mutational hotspot in the wild-type strain may reflect the high frequency of closely opposed lesions. To explain the other mutational hotspots, we propose that the repair of UV lesions is impaired due to the local conformation of the DNA, which might deviate from the B-form. This hypothesis is supported by the excess of mutational hotspots in pyrimidine runs in the Uvr + strain compared to Uvr-. Runs of pyrimidines thus represent both damage- and mutation-prone sequences following UV treatment. , Present address: University of Victoria, Department of Biology, Victoria, B.C., V8W 2Y2, Canada. Correspondence: Dr. Evelyne Sage (present address), Institut Curie, Section de Biologie, 26 rue d'UIm, 75231 Paris Cedex 05, France. Tel. 33 l 40 51 67 01; Fax 33 1 46 33 30 16. Abbreciations: "AA" photolesion, 8,8-adenine dehydrodimer photoproduct; AP site, apurinic/apyrimidinic site; bp, base pair(s); (6-4) Photoproduct, pyrimidine(6-4)pyrimidone pho- toproduct; lay < > Py, cyclobutane pyrimidine dimer; T < > T, cyclobutane thymine dimer; WT, wild-type. The mechanisms whereby DNA damage is transformed into genetic alterations have re- ceived much attention. One approach towards unravelling the molecular mechanisms of muta- tion is to establish mutational spectra. The deter- mination of the chemical nature of DNA damage, its effect on DNA structure, and its distribution in target genes represent a second level of investi- gation. The behaviour of DNA polymerase wi~en it encounters such lesions, and the role of DNA repair in mutagenesis, provide yet other impor- tant considerations (Echols and Goodman, 1990).