The effect of DNA on the activity of human O6-alkylguanine-DNA alkyltransferase was investigated by using O6-benzylguanine as a substrate or inhibitor. The sensitivity of the alkyltransferase to inactivation by O6-benzylguanine was increased by addition of calf thymus DNA. In order to investigate this phenomenon in more detail, the ability of the alkyltransferase to convert O6-benzyl[8-3H]guanine to [8-;3H] guanine 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 absence of DNA. When a mutant P140A alkyltransferase, which is known to be less sensitive to inactivation by O6-benzylguanine presumably as a result of steric hindrance, was used, the rate of reaction was increased by a considerably larger amount, about 16-fold. Oligodeoxynucleotides were able to stimulate the production of guanine from O6-benzylguanine. Single–stranded oligodeoxynucleotides were as effective as double-stranded, and a maximal stimulation was obtained with a 12–mer. These results demonstrate that the alkyltransferase binds to a region of DNA covering at most 12 bases and undergoes a conformational change which facilitates the reaction of adducts at the O6-pposition of guanine with the cysteine acceptor site on the protein. When O6-benzyl[8-3H]deoxyguanosine was used as a substrate, the addition of DNA decreased the rate of formation of 2′deoxy[8-3H]guanosine. Inactivation of the alkyltransferase by O6-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 activation and that the presence of DNA either interferes with the ability to bind O6-benzyldeoxyguanosine or does not favor the reaction with this substrate. These results may explain why O6-benzylguanine is a better inactivator of cellular alkyltransferase than its deoxynucleoside.
All Science Journal Classification (ASJC) codes