Reaction of O6-benzylguanine-resistant mutants of human O6-alkylguanine-DNA alkyltransferase with O6-benzylguanine in oligodeoxyribonucleotides.
Inactivation of the human DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), by O6-benzylguanine renders tumor cells susceptible to killing by alkylating agents. AGT mutants resistant to O6-benzylguanine can be made by converting Pro140 to an alanine (P140A) or Gly156 to an alanine (G156A). These mutations had a much smaller effect on the reaction with O6-benzylguanine when it was incorporated into a short single-stranded oligodeoxyribonucleotide. Such oligodeoxyribonucleotides could form the basis for the design of improved AGT inhibitors. AGT and mutants P140A and G156A preferentially reacted with O6-benzylguanine when incubated with a mixture of two 16-mer oligodeoxyribonucleotides, one containing O6-benzylguanine and the other, O6-methylguanine. When the 6 amino acids located in positions 159-164 in AGT were replaced by the equivalent sequence from the Escherichia coli Ada-C protein (mutant AGT/6ada) the preference for benzyl repair was eliminated. Further mutation incorporating the P140A change into AGT/6ada giving mutant P140A/6ada led to a protein that resembled Ada-C in preference for the repair of methyl groups, but P140A/6ada did not differ from P140A in reaction with the free base O6-benzylguanine. Changes in the AGT active site pocket can therefore affect the preference for repair of O6-benzyl or -methyl groups when present in an oligodeoxyribonucleotide without altering the reaction with free O6-benzylguanine.
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Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033-0850, USA. aep@psu.edu
Kanugula
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Edara
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Pauly
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Moschel
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Goodtzova
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry* / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D017354:Point Mutation
D009838:Oligodeoxyribonucleotides; C064976:O(6)-benzylguanine; D006147:Guanine; D019853:O(6)-Methylguanine-DNA Methyltransferase