Michael J. Horsfall

Missense mutation in the lacI gene of Escherichia coli: Inferences on the structure of the repressor protein

The lac repressor has been studied extensively but a precise three-dimensional structure remains unknown. Studies using mutational data can complement other information and provide insight into protein structure. We have been using the lacI gene-repressor protein system to study the mutational specificity of spontaneous and induced mutation. The sequencing of over 6000 lacI- mutations has revealed 193 missense mutations generating 189 amino acid replacements at 102 different sites within the lac repressor. Replacement sites are not distributed evenly throughout the protein, but are clustered in defined regions. Almost 40% of all sites and over one-half of all substitutions found occur within the amino-terminal 59 amino acid residues, which constitute the DNA-binding domain. The core domain (residues 60 to 360) is less sensitive to amino acid replacement. Here, substitution is found in regions involved in subunit aggregation and at sites surrounding residues that are implicated in sugar-binding. The distribution and nature of missense mutational sites directs attention to particular amino acid residues and residue stretches.

Defect in excision repair alters the mutational specificity of PUVA treatment in the lacI gene of Escherichia coli.

The sequences of 152 lacI- mutations obtained following exposure of Escherichia coli UvrB- strain NR3951 to ultraviolet light in the presence of 8-methoxypsoralen (PUVA treatment) were compared to the spectrum of mutation induced by PUVA treatment in a Uvr+ strain, NR3835. Mutations recovered following PUVA treatment of the UvrB- strain were quite different from those recovered in the Uvr+ strain. In addition, they occurred at a restricted number of unique sites. For example, A.T----T.A base substitutions at position 141, minus G frameshifts at positions 586/587/588 and deletions of 15 base-pairs from position 78 to 92 accounted for 50% or more of mutations recovered in each of the above mutational classes. This altered mutational specificity was accompanied by a failure to recover mutations frequently identified following PUVA treatment of the Uvr+ strain. These mutations include spontaneous-hotspot frameshifts involving the gain or loss of a tetramer 5-CTGG-3 repeated three times at position 620 to 631; and minus A.T base-pair frameshifts recovered at potential T-T crosslink sites. These results indicate that while crosslinks may play a substantial role in the induction of mutation in the Uvr+ strain, they do not contribute substantially to mutagenesis in the UvrB- strain. In addition, the data also suggest that excision repair may not always occur in an error-free manner.

Spontaneous and 9-aminoacridine-induced frameshift mutagenesis: second-site frameshift mutation within the N-terminal region of thelacI gene ofEscherichia coli

SummaryA novel forward mutational system, based on the acquisition of an Iq-d dominant phenotype from an initial Iq− recessive state, was used to identify second-site frameshift mutation [±1(±3n) events] within the N-terminal region of thelacI gene ofEscherichia coli. The DNA sequences are described of forty-six spontaneous and twenty 9-aminoacridine(9-AA)-induced second site mutations. Although −1 frameshift events dominate both spectra, the nature and site specificity of these events clearly distinguish two mutational distributions. The spontaneous distribution contains two −(A: T) frameshift hotspots; one within a monotonic A5 run (9 occurrences), the other at a 5′-CACAACAAC-3′ sequence (12 occurrences). In contrast 17 of the 20 mutations recovered after 9-AA treatment involve the loss of a G: C pair, 14 of which occur at a single site (5′-CGGGC-3′). The striking specificity of the observed mutational hotspots is of interest since this open genetic target contains similar sequences which were infrequently recovered.

Mutational specificity of thymine deprivation-induced mutation in the lacI gene of Escherichia coli

LacI- mutants obtained following 2 and 6 h of thymine deprivation were cloned and sequenced. The mutational spectra recovered were dissimilar. After 2 h of starvation the majority of mutations were base substitutions, largely G:C----C:G transversions. Frameshift mutations but not deletions were observed. In contrast, following 6 h of starvation, with the exception of the G:C----C:G transversion, all possible base substitutions were recovered. Moreover, several deletions but no frameshift events were observed. The differences in the mutational spectra recovered after the two periods of thymine deprivation highlight the role of altered nucleotide pools and the potential influence of DNA replication and repair mechanisms.