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 alk yltransferase (AGT), by O-6-benzylguanine renders tumor cells suscepti ble to killing by alkylating agents, AGT mutants resistant to O-6-benz ylguanine can be made by converting Pro(140) to an alanine (P140A) or Gly(156) to an alanine (G156A), These mutations had a much smaller eff ect on the reaction with O-6-benzylguanine when it was incorporated in to a short single-stranded oligodeoxyribonucleotide. Such oligodeoxyri bonucleotides could form the basis for the design of improved AGT inhi bitors. AGT and mutants P140A and G156A preferentially reacted with O- 6-benzylguanine when incubated with a mixture of two 16-mer oligodeoxy ribonucleotides, one containing O-6-benzylguanine and the other, O-6-m ethylguanine. When the 6 amino acids located in positions 159-164 in A GT were replaced by the equivalent sequence from the Escherichia coli Ada-C protein (mutant AGT/6ada) the preference for benzyl repair was e liminated. Further mutation incorporating the P140A change into AGT/6a da giving mutant P140A/6ada led to a protein that resembled Ada-C in p reference for the repair of methyl groups, but P140A/6ada did not diff er from P140A in reaction with the free base O-6-benzylguanine. Change s in the AGT active site pocket can therefore affect the preference fo r repair of O-6-benzyl or -methyl groups when present in an oligodeoxy ribonucleotide without altering the reaction with free O-6-benzylguani ne.