Specificity of spontaneous mutation in the lacI gene cloned into bacteriophage M13

Abstract

We have studied the specificity of spontaneous mutation in the lacI gene of Escherichia coli cloned into bacteriophage M13. The comparison of the spectrum of 85 spontaneous mutations with that of the lacI gene carried on an E. coli F′ episome revealed the following characteristics: (i) base substitution was predominant, accounting for 80% of spontaneous events compared with only 11% on the F′ episome; (ii) among the base substitutions, the majority were GC→A:T transitions (86%); (iii) not one mutation recovered on M13 corresponded to a mutation at the spontaneous hotspots seen in the F' spectrum (i.e., neither the addition or deletion of the tetramer 5′-CTGG-3′ at position 620–631 nor the A:T→G:C transition at position +6 of lacO were recovered). The enhanced rate of cytosine deamination in single-stranded DNA, the unique replication mechanism and the refractory nature of single-stranded DNA to excision-repair processes present likely explanations for the observed mutational spectrum.