Specific recognition of O6-methylguanine in DNA by active site mutants of human O6-methylguanine-DNA methyltransferase

Tapas K. Hazra, Rabindra Roy, Tapan Biswas, David T. Giabowski, Anthony Pegg, Sankar Mitra

Research output: Contribution to journalArticle

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Abstract

O6-Methylguanine-DNA methyltransferase (MGMT), a ubiquitous DNA repair protein, acts as a monomer in removing the mutagenic DNA adduct O6- alkylguanine (induced by alkylating carcinogens) via a stoichiometric reaction. The alkyl group is transferred without a cofactor to a specific cysteine acceptor residue of MGMT, Cys-145 in the case of human MGMT, containing 207 amino acid residues and thereby inactivates the protein. As a prelude to the investigation of the reaction mechanism of human MGMT by elucidation of its structure in free and substrate-bound forms via NMR spectroscopy and X-ray crystallography, two types of MGMT mutants were generated and characterized. First, systematic deletion analysis of the protein was carried out to determine the smallest size at which it is active or inactive but forms a stable complex with the substrate and so may be useful for NMR spectroscopic analysis. Deletion of more than 8 or 31 residues from the amino or carboxyl terminus, respectively, led to the loss of both activity and substrate binding. Removal of Arg-9 or Leu-176 and distal residues inactivated the protein, presumably by altering its tertiary structure. On the basis of the criteria of bacterial overexpression and solubility, the mutant MGMT with deletion of 28 residues at the carboxyl terminus should be suitable for NMR studies. In the second approach, we examined mutants at the active site (Cys-145) that retain substrate binding. Inactive C145A and C145S substitution mutants were found to form specific and stable complexes with an O6-methylguanine (m6G)-containing oligonucleotide substrate. Wild type MGMT also formed a similar complex, but only as a transient intermediate. Footprinting studies indicated a strong discriminatory effect of the base adduct on the binding of C145A to substrate DNA; 17-18 nucleotides on the m6G-containing strand and 13-14 nucleotides in the complementary strand spanning the base adduct were protected from DNase I digestion by the mutant protein. These results, as well as the identical protease sensitivity of the wild type and mutant proteins, suggest minimal structural change due to conservative mutations at the active site. Thus, the mutant proteins may be utilized for solving the structure and mechanism of human MGMT.

Original languageEnglish (US)
Pages (from-to)5769-5776
Number of pages8
JournalBiochemistry
Volume36
Issue number19
DOIs
StatePublished - May 13 1997

Fingerprint

Methyltransferases
Catalytic Domain
DNA
Mutant Proteins
Substrates
Proteins
Nucleotides
Nuclear magnetic resonance
O-(6)-methylguanine
DNA Adducts
Deoxyribonuclease I
X Ray Crystallography
Spectroscopic analysis
X ray crystallography
Oligonucleotides
DNA Repair
Carcinogens
Solubility
Cysteine
Digestion

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Hazra, Tapas K. ; Roy, Rabindra ; Biswas, Tapan ; Giabowski, David T. ; Pegg, Anthony ; Mitra, Sankar. / Specific recognition of O6-methylguanine in DNA by active site mutants of human O6-methylguanine-DNA methyltransferase. In: Biochemistry. 1997 ; Vol. 36, No. 19. pp. 5769-5776.
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abstract = "O6-Methylguanine-DNA methyltransferase (MGMT), a ubiquitous DNA repair protein, acts as a monomer in removing the mutagenic DNA adduct O6- alkylguanine (induced by alkylating carcinogens) via a stoichiometric reaction. The alkyl group is transferred without a cofactor to a specific cysteine acceptor residue of MGMT, Cys-145 in the case of human MGMT, containing 207 amino acid residues and thereby inactivates the protein. As a prelude to the investigation of the reaction mechanism of human MGMT by elucidation of its structure in free and substrate-bound forms via NMR spectroscopy and X-ray crystallography, two types of MGMT mutants were generated and characterized. First, systematic deletion analysis of the protein was carried out to determine the smallest size at which it is active or inactive but forms a stable complex with the substrate and so may be useful for NMR spectroscopic analysis. Deletion of more than 8 or 31 residues from the amino or carboxyl terminus, respectively, led to the loss of both activity and substrate binding. Removal of Arg-9 or Leu-176 and distal residues inactivated the protein, presumably by altering its tertiary structure. On the basis of the criteria of bacterial overexpression and solubility, the mutant MGMT with deletion of 28 residues at the carboxyl terminus should be suitable for NMR studies. In the second approach, we examined mutants at the active site (Cys-145) that retain substrate binding. Inactive C145A and C145S substitution mutants were found to form specific and stable complexes with an O6-methylguanine (m6G)-containing oligonucleotide substrate. Wild type MGMT also formed a similar complex, but only as a transient intermediate. Footprinting studies indicated a strong discriminatory effect of the base adduct on the binding of C145A to substrate DNA; 17-18 nucleotides on the m6G-containing strand and 13-14 nucleotides in the complementary strand spanning the base adduct were protected from DNase I digestion by the mutant protein. These results, as well as the identical protease sensitivity of the wild type and mutant proteins, suggest minimal structural change due to conservative mutations at the active site. Thus, the mutant proteins may be utilized for solving the structure and mechanism of human MGMT.",
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Specific recognition of O6-methylguanine in DNA by active site mutants of human O6-methylguanine-DNA methyltransferase. / Hazra, Tapas K.; Roy, Rabindra; Biswas, Tapan; Giabowski, David T.; Pegg, Anthony; Mitra, Sankar.

In: Biochemistry, Vol. 36, No. 19, 13.05.1997, p. 5769-5776.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Specific recognition of O6-methylguanine in DNA by active site mutants of human O6-methylguanine-DNA methyltransferase

AU - Hazra, Tapas K.

AU - Roy, Rabindra

AU - Biswas, Tapan

AU - Giabowski, David T.

AU - Pegg, Anthony

AU - Mitra, Sankar

PY - 1997/5/13

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N2 - O6-Methylguanine-DNA methyltransferase (MGMT), a ubiquitous DNA repair protein, acts as a monomer in removing the mutagenic DNA adduct O6- alkylguanine (induced by alkylating carcinogens) via a stoichiometric reaction. The alkyl group is transferred without a cofactor to a specific cysteine acceptor residue of MGMT, Cys-145 in the case of human MGMT, containing 207 amino acid residues and thereby inactivates the protein. As a prelude to the investigation of the reaction mechanism of human MGMT by elucidation of its structure in free and substrate-bound forms via NMR spectroscopy and X-ray crystallography, two types of MGMT mutants were generated and characterized. First, systematic deletion analysis of the protein was carried out to determine the smallest size at which it is active or inactive but forms a stable complex with the substrate and so may be useful for NMR spectroscopic analysis. Deletion of more than 8 or 31 residues from the amino or carboxyl terminus, respectively, led to the loss of both activity and substrate binding. Removal of Arg-9 or Leu-176 and distal residues inactivated the protein, presumably by altering its tertiary structure. On the basis of the criteria of bacterial overexpression and solubility, the mutant MGMT with deletion of 28 residues at the carboxyl terminus should be suitable for NMR studies. In the second approach, we examined mutants at the active site (Cys-145) that retain substrate binding. Inactive C145A and C145S substitution mutants were found to form specific and stable complexes with an O6-methylguanine (m6G)-containing oligonucleotide substrate. Wild type MGMT also formed a similar complex, but only as a transient intermediate. Footprinting studies indicated a strong discriminatory effect of the base adduct on the binding of C145A to substrate DNA; 17-18 nucleotides on the m6G-containing strand and 13-14 nucleotides in the complementary strand spanning the base adduct were protected from DNase I digestion by the mutant protein. These results, as well as the identical protease sensitivity of the wild type and mutant proteins, suggest minimal structural change due to conservative mutations at the active site. Thus, the mutant proteins may be utilized for solving the structure and mechanism of human MGMT.

AB - O6-Methylguanine-DNA methyltransferase (MGMT), a ubiquitous DNA repair protein, acts as a monomer in removing the mutagenic DNA adduct O6- alkylguanine (induced by alkylating carcinogens) via a stoichiometric reaction. The alkyl group is transferred without a cofactor to a specific cysteine acceptor residue of MGMT, Cys-145 in the case of human MGMT, containing 207 amino acid residues and thereby inactivates the protein. As a prelude to the investigation of the reaction mechanism of human MGMT by elucidation of its structure in free and substrate-bound forms via NMR spectroscopy and X-ray crystallography, two types of MGMT mutants were generated and characterized. First, systematic deletion analysis of the protein was carried out to determine the smallest size at which it is active or inactive but forms a stable complex with the substrate and so may be useful for NMR spectroscopic analysis. Deletion of more than 8 or 31 residues from the amino or carboxyl terminus, respectively, led to the loss of both activity and substrate binding. Removal of Arg-9 or Leu-176 and distal residues inactivated the protein, presumably by altering its tertiary structure. On the basis of the criteria of bacterial overexpression and solubility, the mutant MGMT with deletion of 28 residues at the carboxyl terminus should be suitable for NMR studies. In the second approach, we examined mutants at the active site (Cys-145) that retain substrate binding. Inactive C145A and C145S substitution mutants were found to form specific and stable complexes with an O6-methylguanine (m6G)-containing oligonucleotide substrate. Wild type MGMT also formed a similar complex, but only as a transient intermediate. Footprinting studies indicated a strong discriminatory effect of the base adduct on the binding of C145A to substrate DNA; 17-18 nucleotides on the m6G-containing strand and 13-14 nucleotides in the complementary strand spanning the base adduct were protected from DNase I digestion by the mutant protein. These results, as well as the identical protease sensitivity of the wild type and mutant proteins, suggest minimal structural change due to conservative mutations at the active site. Thus, the mutant proteins may be utilized for solving the structure and mechanism of human MGMT.

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