Characterization of human polymorphic DNA repair methyltransferase

Ryo Inoue, Masako Abe, Yusaku Nakabeppu, Mutsuo Sekiguchi, Teruaki Mori, Tomokazu Suzuki

Research output: Contribution to journalArticle

50 Citations (Scopus)

Abstract

The O6-methylguanine-DNA methyltransferase (MGMT) is a critical defence against alkylation-induced mutagenesis and carcinogenesis. More than a 20-fold interindividual difference in the MGMT activity is known to exist among human cultured fibroblasts. We previously reported three allelic variants of the human MGMT gene, namely V1, V2, and V3. Both V1 and V2 carry amino acid substitutions, Leu84Phe and Trp65Cys, respectively, while V3 has a silent mutation. In order to reveal the pharmacogenetic and ecogenetic significance of polymorphism in the human MGMT gene, we investigated the in-vivo characteristics of V1 and V2 methyltransferase enzyme. Esherichia coil strain KT233 (ogt-, ada-) and mer- HeLa MR cells carrying a V1 sequence exhibited almost the same level of sensitivity against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), as did those with a wild-type sequence. The level of methyltransferase protein in those cells was essentially the same as for the wild-type and V1 samples. On the other hand, E. coli and human cells expressing V2 cDNA showed a significantly reduced level of survival. In these cells, V2 protein was hardly detected, even though mRNA was produced normally. An in-vitro translation experiment revealed that the V2 sequence had the potential to produce methyltransferase protein, as did the wild-type and V1 sequences. There was also evidence for a small amount of V2 protein being produced but rapidly degraded, thus implying that the V2 molecule is unstable in vivo. Using purified recombinant proteins, we estimated the kinetic values of wild-type and variant form of enzymes, which would snpport these views. From these results, we concluded that the wild-type and V1 protein have similar enzymatic and physicochemical properties, while V2 protein is considered to be unstable and rare. (C) 2000 Lippincott Williams and Wilkins.

Original languageEnglish
Pages (from-to)59-66
Number of pages8
JournalPharmacogenetics
Volume10
Issue number1
DOIs
Publication statusPublished - Mar 22 2000

Fingerprint

Methyltransferases
DNA Repair
Protein Methyltransferases
DNA
Proteins
Methylnitronitrosoguanidine
Pharmacogenetics
Alkylation
Enzymes
Amino Acid Substitution
HeLa Cells
Recombinant Proteins
Mutagenesis
Genes
Carcinogenesis
Complementary DNA
Fibroblasts
Escherichia coli
Messenger RNA
Survival

All Science Journal Classification (ASJC) codes

  • Genetics
  • Pharmacology, Toxicology and Pharmaceutics(all)

Cite this

Characterization of human polymorphic DNA repair methyltransferase. / Inoue, Ryo; Abe, Masako; Nakabeppu, Yusaku; Sekiguchi, Mutsuo; Mori, Teruaki; Suzuki, Tomokazu.

In: Pharmacogenetics, Vol. 10, No. 1, 22.03.2000, p. 59-66.

Research output: Contribution to journalArticle

Inoue, Ryo ; Abe, Masako ; Nakabeppu, Yusaku ; Sekiguchi, Mutsuo ; Mori, Teruaki ; Suzuki, Tomokazu. / Characterization of human polymorphic DNA repair methyltransferase. In: Pharmacogenetics. 2000 ; Vol. 10, No. 1. pp. 59-66.
@article{c0bce12e2098438daed5bc808a0e3334,
title = "Characterization of human polymorphic DNA repair methyltransferase",
abstract = "The O6-methylguanine-DNA methyltransferase (MGMT) is a critical defence against alkylation-induced mutagenesis and carcinogenesis. More than a 20-fold interindividual difference in the MGMT activity is known to exist among human cultured fibroblasts. We previously reported three allelic variants of the human MGMT gene, namely V1, V2, and V3. Both V1 and V2 carry amino acid substitutions, Leu84Phe and Trp65Cys, respectively, while V3 has a silent mutation. In order to reveal the pharmacogenetic and ecogenetic significance of polymorphism in the human MGMT gene, we investigated the in-vivo characteristics of V1 and V2 methyltransferase enzyme. Esherichia coil strain KT233 (ogt-, ada-) and mer- HeLa MR cells carrying a V1 sequence exhibited almost the same level of sensitivity against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), as did those with a wild-type sequence. The level of methyltransferase protein in those cells was essentially the same as for the wild-type and V1 samples. On the other hand, E. coli and human cells expressing V2 cDNA showed a significantly reduced level of survival. In these cells, V2 protein was hardly detected, even though mRNA was produced normally. An in-vitro translation experiment revealed that the V2 sequence had the potential to produce methyltransferase protein, as did the wild-type and V1 sequences. There was also evidence for a small amount of V2 protein being produced but rapidly degraded, thus implying that the V2 molecule is unstable in vivo. Using purified recombinant proteins, we estimated the kinetic values of wild-type and variant form of enzymes, which would snpport these views. From these results, we concluded that the wild-type and V1 protein have similar enzymatic and physicochemical properties, while V2 protein is considered to be unstable and rare. (C) 2000 Lippincott Williams and Wilkins.",
author = "Ryo Inoue and Masako Abe and Yusaku Nakabeppu and Mutsuo Sekiguchi and Teruaki Mori and Tomokazu Suzuki",
year = "2000",
month = "3",
day = "22",
doi = "10.1097/00008571-200002000-00008",
language = "English",
volume = "10",
pages = "59--66",
journal = "Pharmacogenetics and Genomics",
issn = "1744-6872",
publisher = "Lippincott Williams and Wilkins",
number = "1",

}

TY - JOUR

T1 - Characterization of human polymorphic DNA repair methyltransferase

AU - Inoue, Ryo

AU - Abe, Masako

AU - Nakabeppu, Yusaku

AU - Sekiguchi, Mutsuo

AU - Mori, Teruaki

AU - Suzuki, Tomokazu

PY - 2000/3/22

Y1 - 2000/3/22

N2 - The O6-methylguanine-DNA methyltransferase (MGMT) is a critical defence against alkylation-induced mutagenesis and carcinogenesis. More than a 20-fold interindividual difference in the MGMT activity is known to exist among human cultured fibroblasts. We previously reported three allelic variants of the human MGMT gene, namely V1, V2, and V3. Both V1 and V2 carry amino acid substitutions, Leu84Phe and Trp65Cys, respectively, while V3 has a silent mutation. In order to reveal the pharmacogenetic and ecogenetic significance of polymorphism in the human MGMT gene, we investigated the in-vivo characteristics of V1 and V2 methyltransferase enzyme. Esherichia coil strain KT233 (ogt-, ada-) and mer- HeLa MR cells carrying a V1 sequence exhibited almost the same level of sensitivity against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), as did those with a wild-type sequence. The level of methyltransferase protein in those cells was essentially the same as for the wild-type and V1 samples. On the other hand, E. coli and human cells expressing V2 cDNA showed a significantly reduced level of survival. In these cells, V2 protein was hardly detected, even though mRNA was produced normally. An in-vitro translation experiment revealed that the V2 sequence had the potential to produce methyltransferase protein, as did the wild-type and V1 sequences. There was also evidence for a small amount of V2 protein being produced but rapidly degraded, thus implying that the V2 molecule is unstable in vivo. Using purified recombinant proteins, we estimated the kinetic values of wild-type and variant form of enzymes, which would snpport these views. From these results, we concluded that the wild-type and V1 protein have similar enzymatic and physicochemical properties, while V2 protein is considered to be unstable and rare. (C) 2000 Lippincott Williams and Wilkins.

AB - The O6-methylguanine-DNA methyltransferase (MGMT) is a critical defence against alkylation-induced mutagenesis and carcinogenesis. More than a 20-fold interindividual difference in the MGMT activity is known to exist among human cultured fibroblasts. We previously reported three allelic variants of the human MGMT gene, namely V1, V2, and V3. Both V1 and V2 carry amino acid substitutions, Leu84Phe and Trp65Cys, respectively, while V3 has a silent mutation. In order to reveal the pharmacogenetic and ecogenetic significance of polymorphism in the human MGMT gene, we investigated the in-vivo characteristics of V1 and V2 methyltransferase enzyme. Esherichia coil strain KT233 (ogt-, ada-) and mer- HeLa MR cells carrying a V1 sequence exhibited almost the same level of sensitivity against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), as did those with a wild-type sequence. The level of methyltransferase protein in those cells was essentially the same as for the wild-type and V1 samples. On the other hand, E. coli and human cells expressing V2 cDNA showed a significantly reduced level of survival. In these cells, V2 protein was hardly detected, even though mRNA was produced normally. An in-vitro translation experiment revealed that the V2 sequence had the potential to produce methyltransferase protein, as did the wild-type and V1 sequences. There was also evidence for a small amount of V2 protein being produced but rapidly degraded, thus implying that the V2 molecule is unstable in vivo. Using purified recombinant proteins, we estimated the kinetic values of wild-type and variant form of enzymes, which would snpport these views. From these results, we concluded that the wild-type and V1 protein have similar enzymatic and physicochemical properties, while V2 protein is considered to be unstable and rare. (C) 2000 Lippincott Williams and Wilkins.

UR - http://www.scopus.com/inward/record.url?scp=0034089822&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034089822&partnerID=8YFLogxK

U2 - 10.1097/00008571-200002000-00008

DO - 10.1097/00008571-200002000-00008

M3 - Article

C2 - 10739173

AN - SCOPUS:0034089822

VL - 10

SP - 59

EP - 66

JO - Pharmacogenetics and Genomics

JF - Pharmacogenetics and Genomics

SN - 1744-6872

IS - 1

ER -