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Spectrum of N4-aminocytidine mutagenesis
T Bessho1, K Matsumoto, A Nomura
1Faculty of Pharmaceutical Sciences, Okayama University, Japan.
Abstract:
N4-Aminocytidine, a nucleoside analog, is a potent mutagen towards phages, bacteria, Drosophila and mammalian cells in culture. In vitro, biochemical studies indicate that this reagent acts by being incorporated into DNA. To elucidate the mechanism of N4-aminocytidine mutagenesis, it is essential to identify the nature of DNA sequence alterations taking place during the mutagenesis. We have analyzed the nucleotide sequence changes in the lac promoter-lacZ alpha region of M13mp2 phage induced by treatment of phage-infected Escherichia coli with N4-aminocytidine. The sequence alterations of DNA samples from 89 mutants of the phage were determined. These mutants had single point mutations, except one mutant, in which a double point mutation was detected. Several hot spots were found: however, there are no apparent relations to particular DNA sequences regarding the locations of these spots. All the mutations are transitions; neither transversions nor deletions/insertions were found. A feature in these transitions is that the A/T to G/C and G/C to A/T changes occur at approximately equal rates. The overall picture of the mutagenesis is consistent with a scheme in which misincorporation and misreplication caused by the modified cytosine structure are the key steps in the DNA replication leading to transitions. Similar nucleotide alterations were found for the mutagenesis induced by an alkylated derivative, N'-methyl-N4-aminocytidine. N4-Aminocytidine also induced reversions of these mutants; both A/T to G/C and G/C to A/T transitions again took place.
Insights
N4-Aminocytidine is a mutagen that causes DNA transitions by misincorporation and misreplication. This study details the specific DNA sequence alterations induced by N4-aminocytidine in M13mp2 phage.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- N4-aminocytidine is a nucleoside analog with mutagenic properties across various organisms.
- Understanding the mechanism of mutagenesis requires identifying DNA sequence alterations.
- Previous studies suggest N4-aminocytidine incorporation into DNA is key to its action.
Purpose of the Study:
- To elucidate the mechanism of N4-aminocytidine mutagenesis.
- To identify the specific DNA sequence alterations induced by N4-aminocytidine.
- To analyze mutation hotspots and types.
Main Methods:
- Analysis of nucleotide sequence changes in the M13mp2 phage lac promoter-lacZ alpha region.
- Induction of mutagenesis in phage-infected Escherichia coli using N4-aminocytidine.
- Sequencing of 89 induced mutants.
Main Results:
- All mutations analyzed were single point mutations, primarily transitions (A/T to G/C and G/C to A/T).
- Approximately equal rates of A/T to G/C and G/C to A/T transitions were observed.
- No transversions, deletions, or insertions were detected; mutation hotspots were identified without clear sequence association.
- Similar transition patterns were observed with an N4-aminocytidine derivative and for induced reversions.
Conclusions:
- N4-aminocytidine mutagenesis primarily results in DNA transitions.
- Misincorporation and misreplication of the modified cytosine structure are likely key steps.
- The findings support a model of transition mutagenesis driven by DNA replication errors.