Abstract
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.
Highlights
Inactivation of the human DNA repair protein, O6alkylguanine-DNA alkyltransferase (AGT), by O6-benzylguanine renders tumor cells susceptible to killing by alkylating agents
Many studies have shown that treatment of tumor cells in culture and of tumors grown as xenografts in nude mice with O6benzylguanine to inactivate AGT greatly enhances the killing by both methylating and chloroethylating agents (4, 8 –10)
It appears that resistance can be brought about by two types of alteration of the active site pocket: a steric effect reducing the size of the space available at the active site and preventing the bulky O6-benzylguanine from binding [16, 19]; or the insertion of a hydrophilic residue into the active site, which discourages the binding of the hydrophobic O6-benzylguanine [17]
Summary
Vol 273, No 18, Issue of May 1, pp. 10863–10867, 1998 Printed in U.S.A. Reaction of O6-Benzylguanine-resistant Mutants of Human O6-Alkylguanine-DNA Alkyltransferase with O6-Benzylguanine in Oligodeoxyribonucleotides*. Production of site-directed mutants in the human AGT based on the comparison of these sequences [15,16,17], and experiments using random mutagenesis [18] showed that several mutations causing single amino acid changes in the AGT were able to greatly reduce the sensitivity to inactivation by O6-benzylguanine It appears that resistance can be brought about by two types of alteration of the active site pocket: a steric effect reducing the size of the space available at the active site and preventing the bulky O6-benzylguanine from binding [16, 19]; or the insertion of a hydrophilic residue into the active site, which discourages the binding of the hydrophobic O6-benzylguanine [17]. These mutations did not alter the preference of AGT for the repair of benzyl groups rather than methyl groups when incubated with a mixture of
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