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Recognition of oxidized abasic sites by repair endonucleases
M Häring1, H Rüdiger, B Demple
1Institute of Pharmacology and Toxicology, University of Würzburg, Germany.
Abstract:
The recognition of 'regular' and 'oxidized' sites of base loss (AP sites) in DNA by various AP endonucleases was compared. Model substrates with regular AP sites (resulting from mere hydrolysis of the glycosylic bond) were produced by damaging bacteriophage PM2 DNA by exposure to low pH; those with AP sites oxidized at the C-4'- and C-1'-position of the sugar moiety by exposure to Fe(III)-bleomycin in the presence of H2O2 and to Cu(II)-phenanthroline in the presence of H2O2 and ethanol, respectively. The results confirmed that AP sites-together with single-strand breaks-are indeed the predominant type of DNA modification in all three cases. For the recognition of 4'-oxidized AP sites, a 400-fold higher concentration of Escherichia coli exonuclease III and between 5-fold and 50-fold higher concentrations of bacteriophage T4 endonuclease V, E. coli endonuclease III and E. coli FPG protein were required than for the recognition of regular AP sites. In contrast, the recognition of 4'-oxidized AP sites by E. coli endonuclease IV was effected by 4-fold lower concentrations than needed for regular AP sites. 1'-oxidized AP sites (generated by activated Cu(II)-phenanthroline) were recognized by endonuclease IV and exonuclease III only slightly (3-fold and 13-fold, respectively) less efficiently than regular AP sites. In contrast, there was virtually no recognition of 1'-oxidized AP sites by the enzymes which cleave at the 3' side of AP sites (T4 endonuclease V, endonuclease III and FPG protein). The described differences were exploited for the analysis of the DNA damage induced by hydroxyl radicals, generated by ionizing radiation or Fe(III)-nitrilotriacetate in the presence of H2O2. The results indicate that both regular and 1'-oxidized AP sites represent only minor fractions of the AP sites induced by hydroxyl radicals.
Insights
Enzymes show varied recognition of regular and oxidized DNA abasic (AP) sites. Oxidized AP sites require higher enzyme concentrations, except for E. coli endonuclease IV, impacting DNA damage analysis.
Area of Science:
- Molecular Biology
- DNA Repair Mechanisms
- Biochemistry
Background:
- Abasic (AP) sites are common DNA lesions resulting from hydrolysis or oxidative damage.
- Various AP endonucleases are involved in recognizing and processing AP sites during DNA repair.
- Oxidative modifications at AP sites can alter their recognition by repair enzymes.
Purpose of the Study:
- To compare the recognition of regular and oxidized AP sites by different AP endonucleases.
- To investigate the impact of specific oxidation sites (C-4' and C-1') on enzyme binding and cleavage.
- To utilize differential enzyme recognition for analyzing hydroxyl radical-induced DNA damage.
Main Methods:
- Model DNA substrates with regular AP sites were generated by low pH hydrolysis of bacteriophage PM2 DNA.
- Oxidized AP sites were created using Fe(III)-bleomycin or Cu(II)-phenanthroline with hydrogen peroxide.
- Enzyme concentrations required for recognizing regular versus oxidized AP sites were quantified for E. coli exonuclease III, T4 endonuclease V, E. coli endonuclease III, E. coli FPG protein, and E. coli endonuclease IV.
Main Results:
- Recognition of 4'-oxidized AP sites required significantly higher concentrations of E. coli exonuclease III, T4 endonuclease V, E. coli endonuclease III, and E. coli FPG protein.
- E. coli endonuclease IV showed increased efficiency in recognizing 4'-oxidized AP sites.
- 1'-oxidized AP sites were poorly recognized by enzymes cleaving at the 3' side (T4 endonuclease V, E. coli endonuclease III, E. coli FPG protein) but moderately recognized by endonuclease IV and exonuclease III.
Conclusions:
- AP site oxidation significantly alters recognition by AP endonucleases, with varying effects depending on the enzyme and oxidation site.
- E. coli endonuclease IV exhibits unique recognition properties for oxidized AP sites.
- The differential recognition can be applied to analyze the types of AP sites generated by hydroxyl radical-induced DNA damage, indicating regular and 1'-oxidized AP sites are minor components.