Reaction of O-6-Benzylguanine-Resistant Mutants of Human O-6-Alkylguanine-Dna Alkyltransferase With O-6-Benzylguanine in Oligodeoxyribonucleotides

Inactivation of the human DNA repair protein, O-6-alkylguanine-DNA alkyltransferase (AGT), by O-6-benzylguanine renders tumor cells susceptible to killing by alkylating agents, AGT mutants resistant to O-6-benzylguanine can be made by converting Pro(140) to an alanine (P140A) or Gly(156) to an alanine (G156A), These mutations had a much smaller effect on the reaction with O-6-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 O-6-benzylguanine when incubated with a mixture of two 16-mer oligodeoxyribonucleotides, one containing O-6-benzylguanine and the other, O-6-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 O-6-benzylguanine. Changes in the AGT active site pocket can therefore affect the preference for repair of O-6-benzyl or -methyl groups when present in an oligodeoxyribonucleotide without altering the reaction with free O-6-benzylguanine. [References: 40]

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