C-terminally truncated human O6-alkylguanine-DNA alkyltransferase retains activity.
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 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.
Elder
R H
RH
CRC Department of Carcinogenesis, Paterson Institute for Cancer Research, Christie Hospital (NHS) Trust, Manchester, U.K.
Tumelty
J
J
Douglas
K T
KT
Margison
G P
GP
Rafferty
J A
JA
D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008745:Methylation; D008770:Methylnitrosourea / Q000494:pharmacology; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D019853:O(6)-Methylguanine-DNA Methyltransferase; D010446:Peptide Fragments / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014169:Transformation, Bacterial
D010446:Peptide Fragments; D008770:Methylnitrosourea; D004247:DNA; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; C024408:endodeoxyribonuclease SacI; D015252:Deoxyribonucleases, Type II Site-Specific