Da. Wink et J. Laval, THE FPG PROTEIN, A DNA-REPAIR ENZYME, IS INHIBITED BY THE BIOMEDIATORNITRIC-OXIDE IN-VITRO AND IN-VIVO, Carcinogenesis, 15(10), 1994, pp. 2125-2129
Nitric oxide has been shown to be a mediator molecule in the regulatio
n of many physiological functions. However, this small diatomic molecu
le in the presence of O-2 generates reactive intermediates which modif
y 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 predic
table manner, caused irreversible damage at physiological concentratio
ns to the zinc finger-containing DNA repair enzyme formamidopyrimidine
-DNA glycolyase (Fpg protein). The inhibition of the enzyme activity w
as DEA/NO dose and time dependent with IC(50)s with respect to total N
O released from this compound of approximate to 110 and approximate to
120 mu mol/l respectively. This inhibitory effect by NO was not rever
sible over time in the presence of reducing agents and/or Zn2+. Nitrit
e and diethylamine, the nitrogenous products of the decomposition of D
EA/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 ab
ate the inhibition of the Fpg protein by DEA/NO. Other DNA glycosylase
s 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 inter
action. Our studies were then extended to intact cells. The Fpg protei
n activity was decreased following treatment in vivo when Escherichia
coil MH321 (acr A(-)) cells were treated with DEA/NO. Furthermore, the
Fapy-DNA glycosylase activity in H4 cells, a rat hepatoma line, was d
ecreased when intact cells were incubated with DEA/NO.