A comparative study of the biochemical properties of human and mouse recombinant O6-methylguanine-DNA methyltransferases

R. Roy, S. Shiota, Stephen Kennel, R. Raha, M. Von Wronski, T. P. Brent, S. Mitra

Research output: Contribution to journalArticle

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Abstract

The O6-methylguanine-DNA methyltransferase (MGMT) repairs mutagenic and carcinogenic O6-alkylguanine in DNA by accepting stoichiometrically the alkyl group from the base. Although the mouse MGMT is larger than the human protein because of an additional tetrapeptide sequence, these proteins are 70% homologous. Recombinant MGMTs of the human, the mouse and a mouse mutant with the tetrapeptide deleted were purified to homogeneity from Escherichia coli. The N-terminal amino acid sequences of these proteins are identical to those predicted from the nucleotide sequences, and their molecular masses deter mined by SDS-PAGE agreed with the predicted values. However, the observed isoelectric points of 9.3, 9.2 and 9.3, for the human, mouse and mutant mouse proteins respectively were significantly different from the values, 8.09, 7.47 and 7.49 calculated from the amino acid composition. The extinction coefficients E1%280 nm of human, mouse and mutant mouse protein were calculated from amino acid composition to be 18.2, 11.1 and 11.3 respectively. These values agree fairly well with calculated values. Human and wild-type mouse MGMTs react with the alkylated base in a synthetic DNA substrate poly(dC, dG, m6dG) with comparable second-order rate constants of 2.2×108 and 3.7×108 1/M/min at 37°C respectively and were inactivated by O6-benzylguanine at similar rates. The initial reaction rate (Kin) and rate of inactivation (kinact) constants for reaction with the base were calculated to be 1.8×10-4 M and 1.4×10-3/s for the human protein, 2.3×10-4 M and 1.1×10-3/s for the wild-type mouse protein, and 2.1×10-4 and 1.4×10-3/s for the mutant mouse protein respectively. The MGMTs were inactivated to the extent of 55-65% after heating at 50°C in 20mM Tris-HCI, pH 8.0, 1 mM EDTA, 1 mM DTT and 10% glycerol. However, in the presence of DNA (200 μg/ml), only 25-35% of the protein was inactivated. Both DNA and RNA inhibited all three enzymes in a concentration-dependent fashion, although DNA was a better inhibitor than RNA. High salt (0.2 M NaCl) inhibited human MGMT by 80%, while the wild-type and the mutant mouse MGMTs were inhibited by 55%. The human protein had higher affinity for binding to duplex DNAs than the mouse proteins.

Original languageEnglish (US)
Pages (from-to)405-411
Number of pages7
JournalCarcinogenesis
Volume16
Issue number2
DOIs
StatePublished - Feb 1 1995
Externally publishedYes

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Methyltransferases
DNA
Proteins
Mutant Proteins
O-(6)-methylguanine
RNA
Amino Acids
Isoelectric Point
Edetic Acid
DNA Repair
Glycerol
Heating
Polyacrylamide Gel Electrophoresis
Amino Acid Sequence
Salts
Escherichia coli

All Science Journal Classification (ASJC) codes

  • Cancer Research

Cite this

A comparative study of the biochemical properties of human and mouse recombinant O6-methylguanine-DNA methyltransferases. / Roy, R.; Shiota, S.; Kennel, Stephen; Raha, R.; Von Wronski, M.; Brent, T. P.; Mitra, S.

In: Carcinogenesis, Vol. 16, No. 2, 01.02.1995, p. 405-411.

Research output: Contribution to journalArticle

Roy, R. ; Shiota, S. ; Kennel, Stephen ; Raha, R. ; Von Wronski, M. ; Brent, T. P. ; Mitra, S. / A comparative study of the biochemical properties of human and mouse recombinant O6-methylguanine-DNA methyltransferases. In: Carcinogenesis. 1995 ; Vol. 16, No. 2. pp. 405-411.
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