Large-scale mutational analysis of EMS-induced mutation in the lacI gene of Escherichia coli

Abstract

Mutational spectra produced by mutagens in various repair backgrounds can provide important information about the roles of different repair systems in the mutagenic process. Until recently, such studies have been restricted to the characterisation of comparatively small numbers of mutants or reversion analysis at relatively few sites. The colony hybridisation method used in this study in conjunction with DNA sequencing allows the characterisation of large numbers of mutants and therefore allows analysis of resultant mutational distributions to be made with confidence. We have determined the DNA alterations recovered after treatment with EMS in the N-terminal region of the lacI gene of E. coli. A total of 1138 and 1102 independent lacI-d mutants were characterised in Uvr+ and UvrB-, respectively. Consistent with the known ethylating ability of this compound, the predominant mutation was G:C-->A:T transitions, which accounted for 97% and 93% in Uvr+ and UvrB- strains, respectively. An analysis of the DNA context of mutation induction indicates differential reparability by the Uvr repair pathway. Excision repair appears to more efficiently counter EMS-induced G:C-->A:T transitions at sites flanked by A:T base pairs. However, the influence of excision repair on the ultimate distribution of mutation can not be easily defined with respect to neighbouring sequence.