Structural and immunological comparison of indigenous human O6-methylguanine-DNA methyltransferase with that encoded by a cloned cDNA.
Article Details
- CitationCopy to clipboard
von Wronski MA, Shiota S, Tano K, Mitra S, Bigner DD, Brent TP
Structural and immunological comparison of indigenous human O6-methylguanine-DNA methyltransferase with that encoded by a cloned cDNA.
J Biol Chem. 1991 Jan 15;266(2):1064-70.
- PubMed ID
- 1985934 [ View in PubMed]
- 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.