C-terminally truncated human O6-alkylguanine-DNA alkyltransferase retains activity.
AffiliationCRC Department of Carcinogenesis, Paterson Institute for Cancer Research, Christie Hospital (NHS) Trust, Manchester, U.K.
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AbstractA cDNA encoding the human O6-alkylguanine-DNA alkyltransferase (ATase; EC 188.8.131.52; methylated-DNA: protein-cysteine methyltransferase) has been manipulated to generate a C-terminally deleted protein which retains full methyl-transfer activity. The elimination of 22 amino-acid residues from the C-terminus was achieved by endonuclease-SacI digestion of the 623 bp cDNA coding sequence and ligation of a SacI/HindIII linker containing an in-frame stop codon. The truncated protein was characterized by its reduced molecular mass in immunoblots probed with an antiserum against the full-length protein and by fluorography after incubation with [3H]methylated calf thymus DNA. The rate of methyl transfer was virtually identical for the full-length and truncated ATases. The construction of such a truncated, yet still functional, ATase, with a molecular mass of 19.7 kDa should facilitate a detailed n.m.r. structural study and help to determine the functional significance of the C-terminal domain of mammalian ATases.
CitationC-terminally truncated human O6-alkylguanine-DNA alkyltransferase retains activity. 1992, 285 ( Pt 3):707-9 Biochem. J.
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