Biomaterial Database

Mutations in human O6-alkylguanine-DNA alkyltransferase imparting resistance to O6-benzylguanine. 1994

T M Crone, and K Goodtzova, and S Edara, and A E Pegg

Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey 17033.

O6-Benzylguanine is an inactivator of O6-alkylguanine-DNA alkyltransferases (AGT) which is currently entering clinical trials as an agent improving the cancer chemotherapeutic activity of chloroethylnitrosoureas and other alkylating agents. O6-Benzylguanine acts by virtue of its ability to serve as a substrate for the AGT forming S-benzylcysteine at the cysteine acceptor site. The effects of a number of mutations in the human AGT sequence on the reaction with O6-benzylguanine were investigated by two methods: (a) by measuring the loss of the ability of the AGT to repair a methylated DNA substrate after preincubation with O6-benzylguanine; and (b) by measuring the production of guanine from O6-benzylguanine by the AGT proteins. Both assays gave similar results and showed that mutations of the proline residues at positions 138 and 140 and of the glycine residue at position 156 significantly reduced the ability to react with O6-benzylguanine. The combination of these mutations gave even greater resistance. Thus, the 50% effective dose for O6-benzylguanine was increased from 0.25 microM in the control AGT to 29 microM by mutations P138K/P140A, to 60 microM by mutation G156A and to > 300 microM by mutations P140A/G156A. Truncation of the AGT at the carboxyl end, removing either 31 or 23 amino acids did not affect the activity or the ability to react with O6-benzylguanine, but removal of the 36 carboxyl terminal amino acids, which includes a highly conserved glutamic acid residue, led to the loss of all activity. The rate of the reaction between the AGT and O6-benzylguanine was increased when DNA was present. This increase amounted to about 6-fold with the control AGT and the carboxyl-truncated mutants but was reduced to only 2-fold with G156A mutant and increased to 11-18-fold with the mutations of proline residues at 138 and 140. These results indicate that several residues in the AGT sequence affect the access of the active site to O6-benzylguanine and that these residues are located in at least two regions on either side of the active site cysteine, which is located at residue 145. Mutations in these regions may occur during therapy with alkylating agents and O6-benzylguanine. The development of other AGT inactivators which are still able to inactivate the resistant mutants may be necessary to maximize the potential of AGT inhibition for cancer chemotherapy.

UI	MeSH Term	Description	Entries
D008780	Methyltransferases	A subclass of enzymes of the transferase class that catalyze the transfer of a methyl group from one compound to another. (Dorland, 28th ed) EC 2.1.1.	Methyltransferase
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009154	Mutation	Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations.	Mutations
D004247	DNA	A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine).	DNA, Double-Stranded,Deoxyribonucleic Acid,ds-DNA,DNA, Double Stranded,Double-Stranded DNA,ds DNA
D004351	Drug Resistance	Diminished or failed response of an organism, disease or tissue to the intended effectiveness of a chemical or drug. It should be differentiated from DRUG TOLERANCE which is the progressive diminution of the susceptibility of a human or animal to the effects of a drug, as a result of continued administration.	Resistance, Drug
D006147	Guanine
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D001483	Base Sequence	The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.	DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D001665	Binding Sites	The parts of a macromolecule that directly participate in its specific combination with another molecule.	Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining