Characterization of the Aldehyde Reactive Probe Reaction with AP-Sites in DNA: Influence of AP-Lyase on Adduct Stability

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Abstract
Alkoxyamines react with the open-chain aldehyde form of AP-sites in DNA to produce open-chain aldehyde oximes. Here we characterize the effect of AP-site cleavage by yeast AP-endonuclease 1 (APN1) or T4 pyrimidine dimer DNA glycosylase/AP-lyase (T4 Pdg) on the efficiency and stability of the alkoxyamine aldehyde reactive probe (ARP) condensation reaction with AP-sites. The results indicate that (1) reaction of ARP with the open-chain aldehyde equilibrium form of the AP-site was less efficient than with the 3 ′-α,β-unsaturated aldehyde produced by T4 Pdg; (2) the dRP moiety was least reactive with ARP; (3) both the AP-site and 3 ′-α,β-unsaturated aldehyde were stable with regard to reaction with ARP over a 30-min incubation period at 37°C; and (4) ARP adducted to the open-chain aldehyde form of the AP-site could be replaced by methoxyamine, but the 3 ′-α,β-unsaturated aldehyde ARP oxime was stable against methoxyamine attack.