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Thymine ring saturation and fragmentation products: lesion bypass, misinsertion and implications for mutagenesis
J Evans1, M Maccabee, Z Hatahet
1University of Vermont, Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, Burlington 05405.
Mutation Research
|May 1, 1993
Summary
DNA damage from free radicals creates thymine glycol and ring-fragmentation products that block polymerases. While thymine glycol is bypassed and mutagenic, ring-fragmentation products are lethal, impacting DNA repair and mutagenesis.
Area of Science:
- DNA repair mechanisms
- Molecular biology
- Biochemistry
Background:
- Free radical interactions with DNA pyrimidines generate various lesions.
- Thymine glycol and ring-fragmentation products (urea, beta-ureidoisobutyric acid) are key examples.
- Understanding lesion processing by DNA polymerases is crucial for DNA stability.
Purpose of the Study:
- To investigate the impact of thymine glycol and pyrimidine ring-fragmentation products on DNA polymerases in vitro and in vivo.
- To determine the bypass efficiency and mutagenic potential of these DNA lesions.
- To correlate in vitro findings with in vivo DNA survival and mutation induction.
Main Methods:
- In vitro studies using DNA polymerases with model DNA lesions (thymine glycol, dihydrothymine, urea glycosides, beta-ureidoisobutyric acid, abasic sites).
- Assessing polymerase blocking and termination site frequency.
- In vivo studies using single-stranded DNA in UV-induced Escherichia coli.
- Measuring DNA survival and mutation induction in f1-K12 hybrid DNA.
Main Results:
- Thymine glycol, urea, beta-ureidoisobutyric acid, and abasic sites strongly block DNA polymerases in vitro, while dihydrothymine does not.
- Thymine glycol appears to code for adenine, facilitating bypass and reactivation in vivo.
- Ring-fragmentation products are lethal lesions with inefficient bypass, but beta-ureidoisobutyric acid shows enhanced survival and is a potent premutagenic lesion.
Conclusions:
- In vitro polymerase blocking accurately predicts in vivo lethality for most lesions.
- Thymine glycol is bypassed and can be reactivated in vivo, primarily coding for adenine.
- Ring-fragmentation products, particularly beta-ureidoisobutyric acid, are significant premutagenic lesions, with mutagenesis being sequence-context dependent.