Structural and immunological comparison of indigenous human O6-methylguanine-DNA methyltransferase with that encoded by a cloned cDNA.
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.
von Wronski
M A
MA
Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
Shiota
S
S
Tano
K
K
Mitra
S
S
Bigner
D D
DD
Brent
T P
TP
D000595:Amino Acid Sequence; D001345:Autoradiography; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019853:O(6)-Methylguanine-DNA Methyltransferase; D016415:Sequence Alignment
D004247:DNA; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase