C-Terminally truncated human O6-alkylguanine-DNA alkyltransferase retains activity

R. H. Elder; J. Tumelty; T. K. Douglas; G. P. Margison; J. A. Rafferty

doi:10.1042/bj2850707

C-Terminally truncated human O⁶-alkylguanine-DNA alkyltransferase retains activity

R. H. Elder^*, J. Tumelty, T. K. Douglas, G. P. Margison, J. A. Rafferty

^*Corresponding author for this work

The Christie Hospital NHS Foundation Trust

Research output: Contribution to journal › Article › peer-review

Abstract

A cDNA encoding the human O⁶-alkylguanine-DNA alkyltransferase (ATase; EC 2.1.1.63; 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 Sacl/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.

Original language	English
Pages (from-to)	707-709
Number of pages	3
Journal	Biochemical Journal
Volume	285
Issue number	3
DOIs	https://doi.org/10.1042/bj2850707
Publication status	Published - 1 Aug 1992

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title = "C-Terminally truncated human O6-alkylguanine-DNA alkyltransferase retains activity",

abstract = "A cDNA encoding the human O6-alkylguanine-DNA alkyltransferase (ATase; EC 2.1.1.63; 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 Sacl/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.",

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T1 - C-Terminally truncated human O6-alkylguanine-DNA alkyltransferase retains activity

AU - Elder, R. H.

AU - Tumelty, J.

AU - Douglas, T. K.

AU - Margison, G. P.

AU - Rafferty, J. A.

PY - 1992/8/1

Y1 - 1992/8/1

N2 - A cDNA encoding the human O6-alkylguanine-DNA alkyltransferase (ATase; EC 2.1.1.63; 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 Sacl/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.

AB - A cDNA encoding the human O6-alkylguanine-DNA alkyltransferase (ATase; EC 2.1.1.63; 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 Sacl/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.

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ER -

C-Terminally truncated human O⁶-alkylguanine-DNA alkyltransferase retains activity

Abstract

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