title: Influence of DNA repair by ada and ogt alkyltransferases 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.
authors.affiliation: Departamento de Genética, Facultad de Ciencias, Universidad de Córdoba, Spain.
authors.forename: T
authors.initials: T
authors.lastname: Roldán-Arjona
authors.forename: F L
authors.initials: FL
authors.lastname: Luque-Romero
authors.forename: R R
authors.initials: RR
authors.lastname: Ariza
authors.forename: J
authors.initials: J
authors.lastname: Jurado
authors.forename: C
authors.initials: C
authors.lastname: Pueyo
mesh_terms: D000477:Alkylating Agents / Q000737:chemistry*; D001426:Bacterial Proteins / Q000378:metabolism*; D001482:Base Composition; D004260:DNA Repair; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D008780:Methyltransferases / Q000378:metabolism*; D016296:Mutagenesis; D019853:O(6)-Methylguanine-DNA Methyltransferase; D017354:Point Mutation; D014157:Transcription Factors
chemical_list: D000477:Alkylating Agents; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D014157:Transcription Factors; D008780:Methyltransferases; C045675:Ada protein, E coli; D019853:O(6)-Methylguanine-DNA Methyltransferase; C067511:Ogt protein, E coli