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, Karina Goodtzova

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)10863-10867
Number of pages5
JournalJournal of Biological Chemistry
Volume273
Issue number18
DOIs
StatePublished - May 1 1998

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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