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In vivo mutagenesis by O6-methylguanine built into a unique site in a viral genome
Abstract:
The mutagenicity of O6-methylguanine (O6MeGua), a chemical carcinogen-DNA adduct, has been studied in vivo by using a single-stranded M13mp8 genome in which a single O6MeGua residue was positioned in the unique recognition site for the restriction endonuclease Pst I. Transformation of Escherichia coli MM294A cells with this vector gave progeny phage, of which 0.4% were mutated in their Pst I site. In a separate experiment, cellular levels of O6MeGua-DNA methyltransferase (an O6MeGua-repair protein) were depleted by treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) prior to viral DNA uptake. In these cells, the mutation frequency due to O6MeGua increased with increasing MNNG dose (the highest mutation frequency observed was 20%). DNA sequence analysis of 60 mutant genomes revealed that O6MeGua induced exclusively G-to-A transitions.
Insights
O6-methylguanine (O6MeGua), a DNA adduct, causes mutations. Depleting repair proteins increased O6MeGua mutagenicity, leading exclusively to G-to-A transitions in DNA.
Area of Science:
- Molecular Biology
- Genetics
- Toxicology
Background:
- O6-methylguanine (O6MeGua) is a DNA adduct formed by chemical carcinogens.
- DNA adducts can lead to mutations and potentially cancer.
- Cellular mechanisms exist to repair DNA damage, including O6MeGua.
Purpose of the Study:
- To investigate the mutagenicity of O6MeGua in vivo.
- To determine the effect of DNA repair protein levels on O6MeGua-induced mutations.
- To characterize the types of mutations induced by O6MeGua.
Main Methods:
- Utilized a single-stranded M13mp8 genome with a site-specific O6MeGua adduct.
- Transformed Escherichia coli cells with the modified M13mp8 vector.
- Depleted cellular O6MeGua-DNA methyltransferase levels using N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).
- Analyzed progeny phage for mutations in the Pst I recognition site.
- Performed DNA sequence analysis on mutant genomes.
Main Results:
- A low frequency (0.4%) of mutations in the Pst I site was observed in normal cells.
- Depletion of O6MeGua-DNA methyltransferase significantly increased mutation frequency (up to 20%) in an MNNG dose-dependent manner.
- DNA sequence analysis revealed that O6MeGua exclusively induced G-to-A transitions.
Conclusions:
- O6MeGua is mutagenic in vivo.
- Reduced DNA repair capacity enhances the mutagenic potential of O6MeGua.
- O6MeGua specifically causes G-to-A transitions, indicating a particular replication error mechanism.