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-benzyl-guanine renders tumor cells susceptible to killing by alkylating agents. AGT mutants resistant to O⁶-benzyl-guanine can be made by converting Pro¹⁴⁰ to an alanine (P140A) or Gly¹⁵⁸ 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.