Recognition of oxidized abasic sites by repair endonucleases

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.