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Fapyadenine is a moderately efficient chain terminator for prokaryotic DNA polymerases
M A Graziewicz1, T H Zastawny, R Oliński
1Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw.
Free Radical Biology & Medicine
|February 3, 2000
Summary
Oxidative DNA damage, including FapyAde, can block DNA synthesis. Different DNA polymerases exhibit varying responses to these lesions, suggesting FapyAde is a moderately toxic DNA lesion.
Area of Science:
- Molecular Biology
- Biochemistry
- DNA Repair
Background:
- Oxidative stress can lead to DNA base modifications.
- Understanding how these modifications affect DNA replication is crucial for cellular health.
Purpose of the Study:
- To investigate the impact of oxidative DNA modifications on DNA synthesis by different polymerases.
- To characterize the role of specific lesions, like FapyAde, in DNA replication fidelity and polymerase blockage.
Main Methods:
- Oxidation of single-stranded M13 mp18 DNA using hypoxanthine/xanthine oxidase/Fe3+/EDTA.
- Gas chromatography/isotope dilution mass spectrometry (GC/IDMS-SIM) for identifying modified bases.
- In vitro DNA synthesis assays using T7 DNA polymerase, Klenow fragment, and calf thymus DNA polymerase beta on modified templates.
Main Results:
- Dominant modifications included FapyGua, FapyAde, 8-hydroxyguanine, and thymine glycol.
- Oxidized pyrimidines blocked T7 and Klenow polymerases, while modified purines blocked DNA polymerase beta.
- Increased FapyAde in poly(dA) templates led to premature termination bands during T7 polymerase synthesis.
- Excision of FapyAde, FapyGua, and 8OHGua by Fpg protein revealed or enhanced chain terminations at adenine and guanine sites.
Conclusions:
- FapyAde is a moderately toxic DNA lesion that can arrest DNA synthesis.
- The effect of FapyAde on DNA synthesis is sequence-context and DNA polymerase dependent.
- FapyGua may exhibit similar DNA damaging properties.