Characterization of the aldehyde reactive probe reaction with AP-sites in DNA: influence of AP-lyase on adduct stability.
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'-alpha,beta-unsaturated aldehyde produced by T4 Pdg; (2) the dRP moiety was least reactive with ARP; (3) both the AP-site and 3'-alpha,beta-unsaturated aldehyde were stable with regard to reaction with ARP over a 30-min incubation period at 37 degrees C; and (4) ARP adducted to the open-chain aldehyde form of the AP-site could be replaced by methoxyamine, but the 3'-alpha,beta-unsaturated aldehyde ARP oxime was stable against methoxyamine attack.
Bennett
Samuel E
SE
Department of Environmental and Molecular Toxicology, and the Environmental Health Sciences Center, Oregon State University, Corvallis, Oregon 97331-7301, USA. bennetsa@onid.oregonstate.edu
Kitner
Joshua
J
D000447:Aldehydes / Q000737:chemistry*; D001665:Binding Sites / Q000187:drug effects; D004247:DNA / Q000737:chemistry*; D018736:DNA Adducts / Q000737:chemistry; D004249:DNA Damage; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry*; D043223:Deoxyribonuclease IV (Phage T4-Induced) / Q000737:chemistry; D004795:Enzyme Stability; D015335:Molecular Probes / Q000737:chemistry*
D000447:Aldehydes; D018736:DNA Adducts; D015335:Molecular Probes; D004247:DNA; D043223:Deoxyribonuclease IV (Phage T4-Induced); D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase