THE FPG PROTEIN, A DNA-REPAIR ENZYME, IS INHIBITED BY THE BIOMEDIATOR NITRIC-OXIDE IN-VITRO AND IN-VIVO

Nitric oxide has been shown to be a mediator molecule in the regulation of many physiological functions. However, this small diatomic molecule in the presence of O-2 generates reactive intermediates which modify DNA bases and inactive enzymes at high concentrations (100 mu M). We report that NO generated by 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO, Et(2)NN(O)NO-Na+), a compound known to release NO in a predictable manner, caused irreversible damage at physiological concentrations to the zinc finger-containing DNA repair enzyme formamidopyrimidine-DNA glycolyase (Fpg protein). The inhibition of the enzyme activity was DEA/NO dose and time dependent with IC(50)s with respect to total NO released from this compound of approximate to 110 and approximate to 120 mu mol/l respectively. This inhibitory effect by NO was not reversible over time in the presence of reducing agents and/or Zn2+. Nitrite and diethylamine, the nitrogenous products of the decomposition of DEA/NO, did not inhibit the enzyme. The presence of 500 mu g/ml bovine serum albumin did not protect the protein from the inhibitory effects of DEA/NO, however, the presence of 10 mM cysteine did dramatically abate the inhibition of the Fpg protein by DEA/NO. Other DNA glycosylases tested were not inhibited by exposure to these concentrations of NO. These results, together with reports of site-directed mutagenesis of this protein, suggest that the cysteine residues contained within the zinc finger motif of the Fpg protein are the primary sites of NO interaction. Our studies were then extended to intact cells. The Fpg protein activity was decreased following treatment in vivo when Escherichia coli MH321 (acr A(-)) cells were treated with DEA/NO. Furthermore, the Fapy-DNA glycosylase activity in H4 cells, a rat hepatoma line, was decreased when intact cells were incubated with DEA/NO.