Activation of Human O6-Alkylguanine-DNA Alkyltransferase by DNA

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Abstract
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-position 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.(ABSTRACT TRUNCATED AT 250 WORDS)

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