Activation of human O6-alkylguanine-DNA alkyltransferase by DNA.
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)
Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey 17033.
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Goodtzova
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Crone
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Pegg
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004247:DNA / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D012965:Sodium Chloride / Q000494:pharmacology; D013329:Structure-Activity Relationship
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C064976:O(6)-benzylguanine; D012965:Sodium Chloride; D006147:Guanine; D004247:DNA; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase