Effect of DNA on the inactivation of O⁶-alkylguanine-DNA alkyltransferase by 9-substituted O⁶-benzylguanine derivatives

Studies were carried out on the inactivation of pure human O⁶- alkylguanine-DNA alkyltransferase by 9-substituted O⁶-benzylguanine derivatives in the presence and absence of DNA. The addition of DNA increased the rate of inactivation of the alkyltransferase by O⁶-benzylguanine and its 9-methyl derivative but had little effect on the rate of inactivation by the 9-cyanomethyl derivative. In contrast, when O⁶-benzylguanme derivatives with larger 9-substituents such as ribose, 2'-deoxyribose, dihydrotestosterone, or 2-hydroxy-3(isopropoxy)propyl were used, the addition of DNA was strongly inhibitory to the inactivation. In the case of O⁶-benzylguanine, O⁶- benzylguanosine, and O⁶-benzyl-2'-deoxyguanosine, these results were confirmed by directly measuring the rate of formation by the alkyltransferase of guanine, guanosine, or 2'-deoxyguanosine, respectively. The data indicated that the presence of DNA activated the alkyltransferase, rendering it more reactive with O⁶-benzylguanine or O⁶-benzyl-9-methylguanine, but that DNA interferes with the binding of inhibitors with larger 9-substituents, presumably by competing for the same binding site. Since these inactivators readily inactivate alkyltransferase in cells, the amount of cellular alkyltransferase bound to DNA must be small or readily exchangeable with the free form.