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Oxidized, deaminated cytosines are a source of C --> T transitions in vivo
1Division of Toxicology and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Abstract:
The most common base substitution arising from oxidative damage of DNA is a GC --> AT transition. In an effort to determine the oxidized lesion(s) that gives rise to this mutation, the mutagenicity of three oxidized cytosines, 5-hydroxycytosine, 5-hydroxyuracil, and uracil glycol, were investigated in Escherichia coli. An M13 viral genome was constructed to contain a single oxidized cytosine at a specific site. Replication in vivo of the single-stranded genomes yielded mutation frequencies of 0.05%, 83%, and 80% for 5-hydroxycytosine, 5-hydroxyuracil, and uracil glycol, respectively. The predominant mutation observed was C --> T. A model for C --> T oxidative mutagenesis is suggested in which initial cytosine oxidation is followed by deamination to a poorly repaired uracil derivative that is strongly miscoding during replication.
Insights
Oxidative DNA damage commonly causes GC to AT mutations. Researchers studied oxidized cytosines in E. coli, finding 5-hydroxyuracil and uracil glycol strongly induce C to T mutations.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Oxidative damage to DNA is a significant source of genetic mutations.
- GC to AT transitions are the most frequent base substitutions resulting from such damage.
- Identifying specific oxidized DNA lesions responsible for these mutations is crucial for understanding mutagenesis.
Purpose of the Study:
- To investigate the mutagenicity of three oxidized cytosine derivatives: 5-hydroxycytosine, 5-hydroxyuracil, and uracil glycol.
- To determine which oxidized lesion(s) specifically give rise to GC to AT transitions in DNA.
- To elucidate the mechanism of C to T oxidative mutagenesis.
Main Methods:
- Construction of an M13 viral genome containing a single, site-specific oxidized cytosine.
- In vivo replication of single-stranded viral genomes in Escherichia coli.
- Quantification of mutation frequencies and types, particularly C to T transitions.
Main Results:
- 5-hydroxycytosine induced a mutation frequency of 0.05%.
- 5-hydroxyuracil and uracil glycol induced high mutation frequencies of 83% and 80%, respectively.
- The predominant mutation observed across all tested lesions was cytosine (C) to thymine (T).
Conclusions:
- 5-hydroxyuracil and uracil glycol are potent mutagens that strongly induce C to T oxidative mutations.
- A model is proposed where cytosine oxidation is followed by deamination to a uracil derivative, which is miscoded during DNA replication.
- This pathway highlights a significant mechanism for oxidative mutagenesis and DNA repair challenges.