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Mutagenic specificity of imidazole ring-opened 7-methylpurines in M13mp18 phage DNA
B Tudek1, M Graziewicz, O Kazanova
1Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa. tudek@ibbrain.ibb.waw.pl
Abstract:
The most abundant lesion formed in DNA upon modification with methylating agents 7-methylguanine, under alkaline conditions is converted into 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7MeGua). We have previously shown that treatment of dimethylsulfate methylated DNA with NaOH creates mutagenic base derivatives leading to a 60-fold increase in the frequency of A-->G transitions and a 2-3-fold increase of G-->T and G-->C transversions. We have analyzed which lesions lead to these mutations. We compared mutagenic spectra in the lacZ gene of M13mp18 phage DNA modified with dimethylsulfate and NaOH after selective elimination of damaged bases from molecules used for transfection into SOS-induced E. coli. Partial elimination of Fapy-7MeGua from phage DNA performed by its digestion with formamidopyrimidine-DNA glycosylase resulted in a 2-3-fold decrease of G-->T and G-->C transversions. Selective depurination of methylated bases (9 h, 37 degrees C, pH 7.0) resulting in almost complete loss of 7MeAde as demonstrated by HPLC analysis of [3H]MNU alkylated phage DNA used as a probe, caused a dramatic, 9-fold decrease of A-->G transitions. Alkali-catalysed rearrangement of 7MeAde was followed by HPLC analysis of [3H]MNU alkylated poly(A) and poly(dA). After incubation of these oligonucleotides in NaOH, 7MeAde disappeared from both chromatograms, but only in polyA, 2 new peaks migrating with retention time different from that of 1MeAde, 3MeAde or 7MeAde were detected, suggesting formation of two rotameric forms of Fapy-7MeAde as observed for Fapy-7MeGua. Thus the miscoding lesion, giving rise to A-->G transitions derived from 7MeAde was Fapy-7MeAde. Fapy-7MeGua was at least an order of magnitude less mutagenic, but in SOS-induced cells it gave rise to G-->T and G-->C transversions.
Insights
Alkali treatment of methylated DNA forms mutagenic lesions. 7-methyladenine (7MeAde) rearrangement yields Fapy-7MeAde, causing A-->G mutations, while 7-methylguanine (7MeGua) yields Fapy-7MeGua, causing G-->T/C transversions.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Methylating agents create DNA lesions like 7-methylguanine (7MeGua).
- Alkaline conditions convert these lesions into formamidopyrimidine derivatives (Fapy).
- Previous studies linked dimethylsulfate treatment to increased mutation frequencies.
Purpose of the Study:
- To identify specific DNA lesions responsible for mutations induced by methylating agents under alkaline conditions.
- To compare the mutagenicity of Fapy-7MeGua and Fapy-7MeAde.
- To elucidate the mechanisms of base miscoding during DNA replication.
Main Methods:
- Modification of M13mp18 phage DNA with dimethylsulfate and NaOH.
- Selective elimination of damaged bases using enzymatic digestion (formamidopyrimidine-DNA glycosylase) and chemical depurination.
- Transfection of modified DNA into SOS-induced E. coli.
- Analysis of mutagenic spectra in the lacZ gene.
- High-Performance Liquid Chromatography (HPLC) to characterize base modifications.
Main Results:
- Partial removal of Fapy-7MeGua decreased G-->T and G-->C transversions by 2-3 fold.
- Selective depurination of 7-methyladenine (7MeAde) significantly reduced A-->G transitions by 9-fold.
- Alkali treatment of 7MeAde generated two rotameric forms of Fapy-7MeAde.
- Fapy-7MeAde was identified as the primary lesion causing A-->G transitions.
- Fapy-7MeGua was less mutagenic but induced G-->T and G-->C transversions.
Conclusions:
- Fapy-7MeAde is the principal lesion responsible for A-->G transitions derived from 7MeAde.
- Fapy-7MeGua is a less potent mutagen but contributes to G-->T and G-->C transversions.
- These findings clarify the specific roles of DNA base lesions in chemically induced mutagenesis.