Regulation of the capacity for O6-methylguanine removal from DNA in human lymphoblastoid cells studied by cell hybridization.

Ayres, K; Sklar, R.; K, Larson; Lindgren, Valerie; Strauss, Bernard S.

doi:10.1128/mcb.2.8.904

Cited by 12 publications

(6 citation statements)

References 30 publications

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“…First, the intracellular distributions of tRNA MeTase and the repair enzymes are similar-the major fraction of both enzymes is found in the cytoplasm, although both are present in the nucleus (19,32). Second, Ayres et al (33) found no evidence for a diffusible regulator able to activate or repress the MeTase gene in hybrids between Mex+ and Mex-cells. This is compatible with the full expression of both sets of 06MeGua MeTase genes and the subsequent inactivation of a fraction of the enzyme molecules.…”

Section: Discussionmentioning

confidence: 99%

Possible depletion of a DNA repair enzyme in human lymphoma cells by subversive repair.

Karran¹

1985

Proc. Natl. Acad. Sci. U.S.A.

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Mex+ human lymphoma cell lines contain O6-methylguanine-DNA methyltransferase, a DNA repair enzyme that undergoes suicide inactivation on interaction with its substrate. The cells are therefore competent to remove the alkylation lesion O6-methylguanine from their DNA. However, several repair-deficient lymphoma cell lines (Mex-) are also known. It is shown here that Mex+ cells can be converted temporarily to a Mex- phenotype by growth in nontoxic concentrations of free O6-methylguanine. The depletion of methyltransferase activity is not a result of O6-methylguanine incorporation into DNA and subsequent demethylation by the enzyme. It is proposed that O6-methylguanine is mistakenly incorporated into tRNA molecules by means of a post-transcriptional ribosyl transfer reaction. The demethylation of such bases in tRNA has been demonstrated by using bacterial and human DNA repair enzymes. The existence of such a subversive repair of a methylated base in tRNA raises the possibility of competition between DNA and RNA for cellular DNA repair enzymes. Furthermore, it is proposed that the known aberrant methylation of tRNA in certain transformed cells, together with subversive tRNA repair, could account for the Mex- phenotype.

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Section: Discussionmentioning

confidence: 99%

Possible depletion of a DNA repair enzyme in human lymphoma cells by subversive repair.

Karran¹

1985

Proc. Natl. Acad. Sci. U.S.A.

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“…Karyotypically, they consist of two populations, a majority (-90Y0) that has 46 chromosomes and a minority with 48 chromosomes. L33 cells were derived from a female with infectious mononucleosis and have been described by Ayres et al [24]. Currently they maintain the original number of 47 chromosomes.…”

Section: Cells and Plasmidsmentioning

confidence: 99%

N‐methyl‐N‐nitrosourea‐lnduced mutations in a shuttle plasmid replicated in human cells

Sikpi

Waters

Kraemer

et al. 1990

Molecular Carcinogenesis

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The supF gene of the recombinant shuttle plasmid pZ190 (modified pZ189) was used as a target to study the nature of mutations induced by N-methyl-N-nitrosourea (MNU) in human cells. Treatment of the intact plasmid with MNU followed by its replication in human lymphoblastoid cells led to extensive inactivation and no detectable mutations of the plasmid. However, exposure of the supF DNA fragment alone, followed by its ligation into the vector, caused a ten-fold increase in mutant frequency when replicated in O6-methylguanine-DNA methyltransferase-deficient cells (from 0.54 x 10(-3) to 5.8 x 10(-3)) and an 80-fold increase when replicated in cells containing normal levels of the enzyme (from 0.047 x 10(-3) to 3.8 x 10(-3)). About 45% of the mutant plasmid molecules recovered from human cells contained deletions and insertions. Sixty to 70% of the mutant molecules of wild-type size contained a single-base substitution. Most of these changes were of the G.C----A.T type, consistent with the hypothesis that O6-methylguanine is the primary mutagenic adduct induced by MNU. However, the distribution of mutation sites was highly nonrandom; more than half of all mutations were localized at the G.C position 123, and the rest were distributed in about a dozen sites. The high yield of mutations induced in the supF DNA in a host cell whose capacity for the removal of O6-methylguanine far exceeded the amount present in the supF suggests that the repair of damages in extrachromosomal DNA may be inefficient. This is supported by the observation that the yield of mutations in supF transfected into lymphoblastoid cells devoid of repair activity for O6-methylguanine was comparable to that observed with repair-proficient host cells. The present data, together with results of mutations induced in pZ189 by other agents, strongly suggest that one major determinant of mutational hot spots is the structure of the target DNA itself.

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“…Some subclones of human HeLa S cells-e.g., one obtained from the Massachusetts Institute of Technology Cell Culture Center (HeLa M)-are Mex-, while another subclone-obtained from the American Type Culture Collection (HeLa S3)-has the highest level of MGMT (1.8 x 105 molecules per cell) among all mammalian lines tested (10,17). MGMT expression, while predictable in human/human somatic cell hybrids, appears to be controlled by multiple genetic and epigenetic factors in Mex+ human-Mex-rodent hybrid cells (17)(18)(19). Transformation of Mer+ human cells with simian virus 40 frequently induces the Mer-phenotype, which is observed most commonly in tumor lines (9,15,18).…”

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confidence: 99%

Isolation and structural characterization of a cDNA clone encoding the human DNA repair protein for O6-alkylguanine.

Tano

Shiota

Collier

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

235

125

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O6-Methylguanine-DNA methyltransferase (MGMT; DNA-O6-methylguanine:protein-L-cysteine S-methyltransferase, EC 2.1.1.63), a unique DNA repair protein present in most organisms, removes the carcinogenic and mutagenic adduct O6-alkylguanine from DNA by stoichiometrically accepting the alkyl group on a cysteine residue in a suicide reaction. The mammalian protein is highly regulated in both somatic and germ-line cells. In addition, the toxicity of certain alkylating drugs in tumor and normal cells is inversely related to the levels of this protein. The cDNA of the human gene, henceforth named MGMT, has been cloned in an expression vector on the basis of its rescue of a methyltransferase-deficient (ada-) Escherichia coli host. A 22-kDa active methyltransferase encoded entirely by the cDNA contains an amino acid sequence of 61 residues that bears 60-65% similarity with segments of E. coli methyltransferase (products of the ada and ogt genes), which encompass the alkyl-acceptor residues. The human cDNA has no sequence similarity with the ada and ogt genes, due in part to differences in codon usage, and shows no detectable homology with E. coli genomic DNA. However, it hybridizes with distinct restriction fragments of human, mouse, and rat DNAs. The lack of methyltransferase observed in many human cell lines is due to the absence of the MGMT gene or to lack of synthesis and/or stability of its 0.95-kilobase poly(A)+ RNA transcript.

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Regulation of the capacity for O6-methylguanine removal from DNA in human lymphoblastoid cells studied by cell hybridization.

Cited by 12 publications

References 30 publications

Possible depletion of a DNA repair enzyme in human lymphoma cells by subversive repair.

Possible depletion of a DNA repair enzyme in human lymphoma cells by subversive repair.

N‐methyl‐N‐nitrosourea‐lnduced mutations in a shuttle plasmid replicated in human cells

Isolation and structural characterization of a cDNA clone encoding the human DNA repair protein for O6-alkylguanine.

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