Mutagenesis by 8-methoxypsoralen plus near-UV treatment: analysis of specificity in the lacI gene of Escherichia coli

We have studied the specificity of mutation induced by PUVA treatment in the lacI gene of E. coli. Cells were exposed to near UV (≈365 nm) in the presence of 8-methoxypsoralen under conditions yielding about 7% survival and a 10-fold increase in mutation frequency. The cloning and sequencing of 131 mutants recovered following PUVA treatment revealed that almost all classes of mutation including base substitutions, frameshifts and deletions were induced. The distribution of mutations was non-random and a region of the lacI gene was found to be virtually silent for all classes of mutation. Intriguingly, the broad spectrum of mutation is accompanied by the recovery of mutation at two spontaneous hotspots. We observed a 7-fold increase at a frameshift hotspot involving the gain or loss of a tetramer tandemly repeated 3 times at this site and a 23-fold increase at an A:T→G:C transition hotspot located at the +6 position in the lac operator region. However, despite the presence of these spontaneous hotspots, the mutational spectrum recovered following PUVA treatment was unique and a detailed analysis of the different classes of mutations indicates a role for DNA repair of both monoadducts and cross-links in the production of mutation.