TY - JOUR
T1 - Specific amino acid sequences required for O6-methylguanine-DNA methyltransferase activity
T2 - Analyses of three residues at or near the methyl acceptor site
AU - Ling-ling, Chueh
AU - Nakamura, Takanori
AU - Nakatsu, Yoshimichi
AU - Sakumi, Kunihiko
AU - Hayakawa, Hiroshi
AU - Sekiguchi, Mutsuo
N1 - Funding Information:
We extend special thanks to Dr H.Maki and Dr Y.Nakabeppu for pertinent advice, Dr K.Shimizu and Dr Y.Nakabeppu for providing plasmids, N.Kinoshita for technical assistance and M.Ohara for comments on the manuscript. This work was supported by grants from the Ministry of Education, Science and Culture, Japan (61065007 and 03102007).
Copyright:
Copyright 2010 Elsevier B.V., All rights reserved.
PY - 1992/5
Y1 - 1992/5
N2 - O6-Methylguanine-DNA methyltransferase plays an important role in preventing tumor induction. To elucidate the significance of a highly conserved amino acid sequence of methyltransferase protein, amino acid substitutions were introduced by site-directed mutagenesis of cloned cDNA for human methyltransferase and the activity and stability of mutant forms of enzyme were examined. When cysteine-145, to which the methyl transfer occurs, was replaced by other amino acids, all of the mutants isolated showed the methyl-transferase-negative phenotype. From one of the negative mutants, methyltransferase-positive revertants were isolated, all of which carried codons for cysteine. Thus the cysteine residue is essential for acceptance of the methyl group and cannot be replaced by other amino acids. Using this negative and positive selectionprocedure, analyses were extended to other residues near the acceptor site. At the histidine-146 site, four substitutions (phenyialanine, methionine, asparagine and glutamine) exhibited the positive phenotype but the levels of methyltransferaseactivity in these mutants were low. With valine-148 substitutions there were six types of positive revertants, among which mutants carrying isoleucine, cysteine and alanine showed significantly high levels of methyltransferase activity. Some mutant forms of cDNA were expressed in methyltransferase-defkient human cells, and the results obtained with Escherichia coli cells were confirmed.
AB - O6-Methylguanine-DNA methyltransferase plays an important role in preventing tumor induction. To elucidate the significance of a highly conserved amino acid sequence of methyltransferase protein, amino acid substitutions were introduced by site-directed mutagenesis of cloned cDNA for human methyltransferase and the activity and stability of mutant forms of enzyme were examined. When cysteine-145, to which the methyl transfer occurs, was replaced by other amino acids, all of the mutants isolated showed the methyl-transferase-negative phenotype. From one of the negative mutants, methyltransferase-positive revertants were isolated, all of which carried codons for cysteine. Thus the cysteine residue is essential for acceptance of the methyl group and cannot be replaced by other amino acids. Using this negative and positive selectionprocedure, analyses were extended to other residues near the acceptor site. At the histidine-146 site, four substitutions (phenyialanine, methionine, asparagine and glutamine) exhibited the positive phenotype but the levels of methyltransferaseactivity in these mutants were low. With valine-148 substitutions there were six types of positive revertants, among which mutants carrying isoleucine, cysteine and alanine showed significantly high levels of methyltransferase activity. Some mutant forms of cDNA were expressed in methyltransferase-defkient human cells, and the results obtained with Escherichia coli cells were confirmed.
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U2 - 10.1093/carcin/13.5.837
DO - 10.1093/carcin/13.5.837
M3 - Article
C2 - 1586996
AN - SCOPUS:0026589821
VL - 13
SP - 837
EP - 843
JO - Carcinogenesis
JF - Carcinogenesis
SN - 0143-3334
IS - 5
ER -