Influence of DNA repair by ada and ogt alkyltransferases on the mutational specificity of alkylating agents.
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.
Roldán-Arjona
T
T
Departamento de Genética, Facultad de Ciencias, Universidad de Córdoba, Spain.
Luque-Romero
F L
FL
Ariza
R R
RR
Jurado
J
J
Pueyo
C
C
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
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