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J Biol Chem, Vol. 273, Issue 18, 10863-10867, May 1, 1998

Reaction of O⁶-Benzylguanine-resistant Mutants of Human O⁶-Alkylguanine-DNA Alkyltransferase with O⁶-Benzylguanine in Oligodeoxyribonucleotides

Anthony E. Pegg, Sreenivas Kanugula, Suvarchala Edara, Gary T. Pauly^¶, Robert C. Moschel^¶, and Karina Goodtzova

From the  Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033-0850, and ^¶ ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201

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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