ACTIVATION OF HUMAN O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE BY DNA

The effect of DNA on the activity of human O-6-alkylguanine-DNA alkylt ransferase was investigated by using O-6-benzylguanine as a substrate or inhibitor. The sensitivity of the alkyltransferase to inactivation by O-6-benzylguanine was increased by addition of calf thymus DNA. In order to investigate this phenomenon in more detail, the ability of th e alkyltransferase to convert O-6-benzyl[8-H-3]guanine to [8-H-3]guani ne was measured. The rate of guanine production was increased about 6- fold by addition of DNA. The effect of DNA was completely abolished by addition of 0.2 M NaCl, which had no effect on the reaction in the ab sence of DNA. When a mutant P140A alkyltransferase, which is known to be less sensitive to inactivation by O-6-benzylguanine presumably as a result of steric hindrance, was used, the rate of reaction was increa sed by a considerably larger amount, about 16-fold. Oligodeoxynucleoti des were able to stimulate the production of guanine from O-6-benzylgu anine. Single-stranded oligodeoxynucleotides were as effective as doub le-stranded, and a maximal stimulation was obtained with a 12-mer. The se results demonstrate that the alkyltransferase binds to a region of DNA covering at most 12 bases and undergoes a conformational change wh ich facilitates the reaction of adducts at the O-6-position of guanine with the cysteine acceptor site on the protein. When O-6-benzyl[8-H-3 ] deoxyguanosine was used as a substrate, the addition of DNA decrease d the rate of formation of 2'-deoxy[8-H-3]guanosine. Inactivation of t he alkyltransferase by O-6-benzyldeoxyguanosine was also inhibited by DNA addition. This suggests that binding of the deoxynucleoside alone is not sufficient to cause the conformational change needed for activa tion and that the presence of DNA either interferes with the ability t o bind O-6-benzyldeoxyguanosine or does not favor the reaction with th is substrate. These results may explain why O-6-benzylguanine is a bet ter inactivator of cellular alkyltransferase than its deoxynucleoside.