Interaction of DNA containing Fapy.dA or its C-nucleoside analogues with base excision repair enzymes. Implications for mutagenesis and enzyme inhibition

Fapy.dA is produced in DNA as a result of oxidative stress. Recently, this lesion and its C-nucleoside analogues were incorporated in chemically synthesized oligonucleotides at defined sites. The interaction of DNA containing Fapy.dA or nonhydrolyzable analogues with Fpg and MutY is described. Fpg e...

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Bibliographic Details
Published in:Biochemistry (Easton) 2002-12, Vol.41 (52), p.15838
Main Authors: Wiederholt, Carissa J, Delaney, Michael O, Greenberg, Marc M
Format: Article
Language:English
Subjects:
Base Pair Mismatch
Binding Sites
Deoxyadenosines - chemistry
Deoxyadenosines - genetics
Deoxyribonucleosides - chemistry
Deoxyribonucleosides - genetics
DNA - chemistry
DNA Damage
DNA Glycosylases
DNA Repair
DNA-Formamidopyrimidine Glycosylase
Enzyme Inhibitors - chemistry
Enzyme Stability
Mutagenesis
N-Glycosyl Hydrolases - antagonists & inhibitors
N-Glycosyl Hydrolases - chemistry
N-Glycosyl Hydrolases - genetics
Nucleic Acid Denaturation
Nucleic Acid Heteroduplexes - chemistry
Nucleic Acid Heteroduplexes - genetics
Pyrimidines - chemistry
Thermodynamics
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Summary:Fapy.dA is produced in DNA as a result of oxidative stress. Recently, this lesion and its C-nucleoside analogues were incorporated in chemically synthesized oligonucleotides at defined sites. The interaction of DNA containing Fapy.dA or nonhydrolyzable analogues with Fpg and MutY is described. Fpg efficiently excises Fapy.dA (K(m) = 1.2 nM, k(cat) = 0.12 min(-1)) opposite T. The lesion is removed as efficiently from duplexes containing Fapy.dA:dA or Fapy.dA:dG base pairs. Multiple turnovers are observed for the repair of Fapy.dA mispairs in a short period of time, indicating that the enzyme does not remain bound to the product duplex. MutY does not incise dA from a duplex containing this nucleotide opposite Fapy.dA, nor does it exhibit an increased level of binding compared to DNA composed solely of native base pairs. MutY also does not incise Fapy.dA when the lesion is opposite dG. These data suggest that Fapy.dA could be deleterious to the genome. Fpg strongly binds duplexes containing the beta-C-nucleoside analogue of Fapy.dA (beta-C-Fapy.dA) opposite all native nucleotides (K(D) < 27 nM), as well as the alpha-C-nucleoside (alpha-C-Fapy.dA) opposite dC (K(D) = 7.1 +/- 1.5 nM). A duplex containing a beta-C-Fapy.dA:T base pair is an effective inhibitor (K(I) = 3.5 +/- 0.3 nM) of repair of Fapy.dA by Fpg, suggesting the C-nucleoside may have useful therapeutic properties.
ISSN:0006-2960