Structural and immunological comparison of indigenous human O6-methylguanine-DNA methyltransferase with that encoded by a cloned cDNA

M. A. Von Wronski, S. Shiota, K. Tano, S. Mitra, D. D. Bigner, T. P. Brent

Research output: Contribution to journalArticle

22 Scopus citations

Abstract

O6-Methylguanine-DNA methyltransferase, a ubiquitous and unusual DNA repair protein, eliminates mutagenic and cytotoxic O6-alkylguanine from DNA by transferring the alkyl group to one of its cysteine residues in a second-order suicide reaction. This 22-kDa protein was immunoaffinity-purified to homogeneity from cultured human lymphoblasts (CEM-CCRF line) and compared with the O6-methylguanine-DNA methyltransferase purified to homogeneity from Escherichia coli expressing a cloned human cDNA. The cellular and recombinant proteins were identical in size, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of intact molecules and their peptides. Immunoprobing of Western blots with three monoclonal antibodies specific for human cellular O6-methylguanine-DNA methyltransferase further indicated identity of the two proteins. The amino acid sequence of the cellular protein was experimentally determined for 87 out of a total of 207 residues and was found to be identical to that deduced from the cDNA sequence. A unique cysteine residue at position 145 was identified as the methyl acceptor site by autoradiographic analysis of peptides and sequence analysis of 3H-methylated O6-methylguanine-DNA methyltransferase. These observations establish that the cloned O6-methylguanine-DNA methyltransferase cDNA encodes the full-length O6-methylguanine-DNA methyltransferase polypeptide that is normally present in human cells. Moreover, the cellular protein does not appear to be significantly modified by posttranslational processes.

Original languageEnglish (US)
Pages (from-to)1064-1070
Number of pages7
JournalJournal of Biological Chemistry
Volume266
Issue number2
StatePublished - 1991

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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