Influence of DNA repair by ada and ogt alkytransferases on the mutational specificity of alkylating agents

Abstract

We investigated the influence of the alkyltransferases (ATases) encoded by the ada and ogt genes of Escherichia coli on the mutational specificity of alkylating agents. A new mutational assay for selection of supF- mutations in shuttle-vector plasmids was used. Treating plasmid-bearing bacteria with N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), and ethyl methanesulfonate (EMS) dramatically increased the mutation frequency (from 33-fold to 789-fold). The vast majority of mutations (89-100%) were G:C-->A:T transitions. This type of mutation increased in ada- (MNU) or ogt- (ENU) bacteria, suggesting that repair of O6-methylguanine by ada ATase and repair of O6-ethylguanine by ogt ATase contribute mainly to the decrease in G:C-->A:T transitions. The analysis of neighboring base sequences revealed an overabundance of G:C-->A:T transitions at 5'-GG sequences. The 5'-PuG bias increased in ATase-defective cells, suggesting that these sequences were not refractory to repair. G:C-->A:T transitions occurred preferentially in the untranscribed strand after in vivo exposure. That this strand specificity was detected even in bacteria devoid of ATase activity (ada- ogt-) and not after in vitro mutagenesis suggests a bias for damage induction rather than for DNA repair. Highly significant differences were found between the in vivo and in vitro incidences of G:C-->A:T substitutions at the two major hotspots, positions 123 (5'-GGG-3'; antisense strand) and 168 (5'-GGA-3'; sense strand). These results are explained by differences in the probability of formation of stem-loop structures in vivo and in vitro.

Determining mutational fingerprints at the human p53 locus with a yeast functional assay: a new tool for molecular epidemiology

“…At both loci, the incidence and the location of GC4AT transitions are consistent with the hypothesis that O6-alkylguanine is the main determinant of CCNU mutagenicity. Alkylnitrosoureas preferentially cause GC4AT transitions at Gs preceded by a Pu residue in E. coli (Jurado et al, 1995;RoldaÁ n-Arjona et al, 1994;Richardson et al, 1987;Burns et al, 1988) and in eukaryotes (Inga et al, 1995;Kunz and Mis, 1989;Minnick et al, 1992;Moriwaki et al, 1991;Palombo et al, 1992). The 5'-¯anking purine eect has been explained as the consequence of a structural in¯uence on the initial formation of O6-alkylguanine, which may be further enhanced by alkylation speci®c repair (Horsfall et al, 1990).…”

A new approach to random mutagenesis in vitro

“…Our results have demonstrated that it is a very effective way of evolving enzyme/protein in vitro. In comparison to whole plasmid mutagenesis (Roldan-Arjona et al, 1994), our approach has the advantage of eliminating the potential phenotype changes caused by mutations outside the target region, such as mutations resulting in change of plasmid copy numbers and regulatory signals for transcription and translation. An obvious extension of this approach would be to further narrow the target region.…”

Mutation spectra analysis suggests that N-(2-chloroethyl)-N′-cyclohexyl-N-nitrosourea-induced lesions are subject to transcription-coupled repair in Escherichia coli

“…Indeed, all strains we examined had similar incidences of GC→AT transitions ( Table 2) that occurred in similar sequence contexts (more than 96% were preceded 5´ by a purine) ( Table 4). It is known that alkylnitrosoureas preferentially cause GC→AT transitions at Gs preceded by a purine residue in E. coli [9,32,33] and in eukaryotes [1,34,35]. The 5´-flanking purine effect has been explained as the consequence of structural influence on the initial formation of O 6 -chloroethylguanine that may be further enhanced by sequence-specific repair [36].…”