Inhibition of rat liver DNA polymerase by nitrosoureas and isocyanates

The effects of 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexl-1-nitrosourea, and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea on two nonmitochondrial DNA polymerases (I and II) purified from rat liver and hepatoma were examined. The activity of DNA polymerase I was not altered by treatment with any of the nitrosoureas or the corresponding isocyanates, 2-chloroethyl isocyanate and cyclohexyl isocyanate. Incubation of DNA polymerase II with the nitrosoureas (1 mM) inhibited its enzymatic activity 30 to 45%. DNA polymerase II was inhibited 75 and 90% by 1.mM 2-chloroethyl isocyanate and cyclohexyl isocyanate, respectively. The nitrosoureas appear to exert their inhabitory action on the enzyme (DNA polymerase II) rather than on the DNA template. Pretreatment of the enzyme increased the degree of inhibition by 1 mM nitrosourea (50 to 60% inhibition) or 2-chloroethul isocyanate (greater than 90% inhibition), whereas pretreatment of the DNA template did not enhance the inhibitory effect. The three nitrosoureas are equally effective as inhibitors of DNA polymerase II. 2-Chloroethyl isocyanate and cyclohexyl isocyanate are better inhibitors than are the nitrosoureas. Since further decomposition products of the isocyanates, 2-chloroethylamine and cyclohexylamine, do not inhibit DNA polymerase II, we conclude that the isocyanates, which are decomposition products of the nitrosoureas, are the active inhibitors of the enzyme.