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P Karran

Publications and source records attributed to P Karran.

At least 73 records · Page 4Linked to original sources
PubMed1988

Repair of O-alkylpyrimidines in mammalian cells: a present consensus.

Enzymatic repair of the O-alkylpyrimidines (O2- and O4-alkylthymine, O2-alkylcytosine) and alkyl phosphotriesters has been studied in Escherichia coli, and the two proteins involved, a glycosylase (DNA-3-methyladenine glycosylase) and a methyltransferase (DNA-O6-methylguanine:protein-L-cysteine S-methyltransferase, EC 2.1.1.63), have been well characterized. In mammals or mammalian cells treated with carcinogenic alkylating agents, loss of these derivatives has been demonstrated repeatedly. Nevertheless, mammalian repair proteins that are analogous to those from E. coli do not detectably act on these alkyl derivatives. A variety of techniques has been used by many investigators in the United States and Europe, who conclude here that the mode of O-alkylpyrimidine and alkyl phosphotriester repair in mammalian cells differs from that in E. coli. New approaches and methods are needed to characterize these processes at the biochemical and molecular level.

Alkylating Agents↗
PubMed1988

The contribution of O6-methylguanine and methylphosphotriesters to the cytotoxicity of alkylating agents in mammalian cells.

A modified Escherichia coli ada+ gene which encodes methyltransferase active on O6-methylguanine, but not methylphosphotriester residues in DNA has been introduced by transfection into Chinese hamster ovary cells. Expression of the altered Ada protein in these cells conferred resistance to methylating agents. However, in two independently derived cell lines, the relation between enzyme activity and the degree of protection was not quantitative. The molecular nature of the Ada protein fragment produced differed between the two cell lines. In addition, cell lines exhibiting increased resistance to N-methyl-N'-nitro-N-nitrosoguanidine have been derived from a cell line expressing the Ada protein methylphosphotriester repair function. The resistant cell lines expressed elevated levels of methylphosphotriester repair protein. However, this enhanced DNA repair activity was not responsible for the observed resistance.

Alkylating Agents↗
PubMed1986

Complementation of sensitivity to alkylating agents in Escherichia coli and Chinese hamster ovary cells by expression of a cloned bacterial DNA repair gene.

Dual expression vectors derived from pSV2gpt and encoding all or part of the Escherichia coli ada+ gene have been constructed. Following transformation into an E. coli ada strain or transfection and stable integration into the genome of Chinese hamster ovary (CHO) cells, plasmid vectors containing the whole ada+ gene conferred resistance to both killing and mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Thus, the bacterial DNA repair gene was functionally expressed in the mammalian cells. Plasmids containing an N-terminal fragment of the ada+ gene which encoded only one of the two methyltransferase activities of the Ada protein did not significantly protect E. coli or CHO cells against MNNG. These results are consistent with the central role of the intact ada+ gene in controlling the adaptive response to alkylating agents in E. coli. However, the data further suggest that some alkylation lesions in DNA, such as O6-methylguanine, may exert partly different biological effects in E. coli and mammalian cells.

Animals↗
PubMed1986

Site-specific mutagenesis in vivo by single methylated or deaminated purine bases.

The technique of site-directed mutagenesis has been used to investigate the mutagenicity of O6-methylguanine (O6-MeG) or hypoxanthine introduced as a single lesion at a specific locus in an M13mp9 RF molecule constructed in vitro. Following transformation of O6-MeG-containing RF molecules into E. coli JM101, mutant progeny phage were produced at a frequency not significantly different from that observed with wild-type M13mp9 RF. The mutant yield was greatly enhanced by exhausting cellular O6-MeG DNA-methyltransferase before transformation. In contrast, hypoxanthine exhibited miscoding mutagenesis in the absence of interference with cellular repair mechanisms. This indicates that cellular hypoxanthine-DNA glycosylase acts inefficiently in the removal of hypoxanthine from DNA in vivo. The precise mutational changes induced by hypoxanthine were determined by DNA sequence analysis.

Base Sequence↗
PubMed1985

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

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.

Burkitt Lymphoma↗
PubMed1985

The cytotoxic and mutagenic effects of alkylating agents on human lymphoid cells are caused by different DNA lesions.

The Burkitt's lymphoma cell line Raji has a Mex+ phenotype. It is more resistant to killing by alkylating agents than a sub line (Raji TK-) which is Mex-. A reduction in O6-methylguanine (O6MeG)-DNA methyltransferase can be brought about by growing Raji cells in the presence of free O6MeG. The depletion in enzyme activity is specific and reversible; removal of O6MeG from the medium results in the restoration of methyltransferase activity within 4 h. Raji cells, in which methyltransferase has been reduced by this treatment to below detectable levels, are not sensitised to killing by N-methyl-N'-nitro-N-nitrosoguanidine or the cross-linking nitrosoureas, 1,3-bis(2-chloroethyl)-1-nitrosourea and 1-(2-chloroethyl)-1-nitrosourea. This implies that adducts at the O6 atom of guanine in DNA are not potentially cytotoxic lesions. Secondly, it suggests that a defect other than the lack of methyltransferase is responsible for the sensitivity of Mex- cells to killing by alkylating agents.

Alkylating Agents↗
PubMed1984

Inducible repair of O-alkylated DNA pyrimidines in Escherichia coli.

The three miscoding alkylated pyrimidines O2-methylcytosine, O2-methylthymine and O4-methylthymine are specifically recognized by Escherichia coli DNA repair enzymes. The activities are induced as part of the adaptive response to alkylating agents. O2-Methylcytosine and O2-methylthymine are removed by a DNA glycosylase, the alkA+ gene product, which also acts on N3-methylated purines. O4-Methylthymine is repaired by a methyltransferase, previously known to correct O6-methylguanine by transfer of the methyl group to one of its own cysteine residues. It is proposed that certain common structural features of the various methylated bases allow each of the two inducible repair enzymes to recognize and remove several different kinds of lesions from alkylated DNA.

Alkylating Agents↗
PubMed1983

A biochemical defect in the repair of alkylated DNA in cells from an immunodeficient patient (46BR).

The fibroblast cell strain 46BR, derived from an immunodeficient individual, is hypersensitive to the lethal effects of a variety of DNA-damaging agents, this effect being particularly marked for monofunctional methylating agents. After u.v. irradiation 46BR cells show normal unscheduled DNA synthesis, daughter strand repair, and recovery of DNA and RNA synthesis. The inhibition of DNA replicative synthesis by u.v. is slightly less than that of normal cells. After gamma-irradiation the rejoining of strand breaks is normal as are the kinetics of replicative DNA synthesis. Following treatment with dimethylsulphate, replicative DNA synthesis is affected in a similar way to normal cells, unscheduled DNA synthesis may be increased relative to normal cells, but more strand breaks persist in 46BR than in normal cells. In addition 46BR cells are hypersensitive to the toxic effects of 3-aminobenzamide, an inhibitor of ADP-ribosyl transferase. This enzyme is involved in the ligation step of repair of alkylation damage. A hypothesis is presented suggesting that 46BR may be defective in DNA ligase I.

Benzamides↗
PubMed1983

O6-Methylguanine-DNA methyltransferase of human lymphoid cells: structural and kinetic properties and absence in repair-deficient cells.

Human lymphoid cell lines contain a DNA repair enzyme which removes the mutagenic alkylation lesion O6-methylguanine from DNA. The enzyme transfers the methyl group to a protein cysteine residue, generating S-methylcysteine, and is inactivated as a consequence of the reaction. Apparently the methylated enzyme represents a dead-end complex. The transfer reaction is very rapid and is completed in less than 1 min at 37 degrees, but methyl group transfer from single-stranded DNA or heavily damaged DNA is less efficient. The active methyltransferase and the methylated protein both have molecular weights of 21,000 to 22,000, as determined by gel filtration. Lymphoid cell lines proficient in repair of O6-methylguanine in vivo, Mex+, contain 10,000 to 25,000 molecules of the methyltransferase per cell. In contrast, repair-deficient cell lines, Mex-, do not contain detectable amounts of the enzyme. The latter point was verified by applying a partial purification procedure for the enzyme to cell-free extracts from two Mex- cell lines.

Burkitt Lymphoma↗
PubMed1982

Excision of O6-methylguanine from DNA by human fibroblasts determined by a sensitive competition method.

A new, simple and relatively inexpensive assay for measuring O6-methylguanine (O6-MeG) in methylated DNA is described. This assay used the property of suicide inactivation of Escherichia coli methyltransferase. When crude extracts of methyltransferase are incubated with DNA containing O6-MeG, transfer of the methyl group to a cysteine residue on the protein occurs and the protein is subsequently inactivated. Each protein molecular has been shown to act only once. In our assay, methylated DNA is first incubated with a fixed amount of the extract. The O6-MeG present in this DNA proportionally inactivates a part of the methyltransferase activity. The remaining activity is then incubated with a fixed amount of O6-[3H]MeG substrate of known specific activity and precipitated with acid. Hydrolysis of the acid precipitable fraction releases the unaltered O6-[3H]MeG enabling the amount of O6-MeG present in the unknown sample to be calculated. This method enables the accurate measurement of as little as 0.1 pmol of O6-MeG in methylated DNA and compares favourably with current radiochemical and immunological techniques. We have used this assay to measure O6-MeG produced in the DNA of human fibroblasts treated with N-methyl-N'-nitro-N-nitrosoguanidine. We have also studied the kinetics of removal of this lesion. We confirm previous reports of a rapid repair system for the removal of O6-MeG from DNA by human fibroblasts.

DNA↗
PubMed1981

Specificity of the bacteriophage PBS2 induced inhibitor of uracil-DNA glycosylase.

The purified PBS2 phage-coded inhibitor of uracil-DNA glycosylase (Ura-DNA glycosylase) from Bacillus subtilis has been tested for its ability to inhibit this enzyme isolated from other prokaryotic and from eukaryotic sources. In addition, the inhibitor has been assayed for its effect on DNA glycosylases specific for other base residues in DNA. The data indicate that Ura-DNA glycosylases from a variety of sources are equally sensitive to inhibition by the inhibitor. DNA glycosylases specific for base residues in DNA other than uracil are not inhibited by the PBS2-coded inhibitor.

Bacillus subtilis↗
PubMed1981

DNA repair.

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Alkylating Agents↗
PubMed1980

Hypoxanthine in deoxyribonucleic acid: generation by heat-induced hydrolysis of adenine residues and release in free form by a deoxyribonucleic acid glycosylase from calf thymus.

A slow conversion of adenine residues to hypoxanthine occurs in single-stranded DNA when heated in neutral aqueous buffers. The rate of this reaction at pH 7.6 and 110 degrees C is k = 4 x 10(-8) s-1, as determined by base analysis of heat-treated DNA that contains radioactively labeled adenine residues. It is proposed that adenine deamination is one of several forms of hydrolytic damage that may occur as spontaneous premutagenic lesions in DNA in vivo. Cell extracts from calf thymus and human fibroblasts contain a DNA glycosylase activity with specifically catalyzes the release of free hypoxanthine from DNA or polydeoxyribonucleotides that contain dIMP residues. Several properties of the purified enzyme from calf thymus are described: It has an approximate molecular weight of 31 000. No cofactors are required for activity. The enzymatic release of hypoxanthine occurs readily from double-stranded polydeoxyribonucleotides that have either thymine or cytosine residues in the complementary strand. Single-stranded polymers are 10-20-fold more slowly attacked, and there is no detectable cleavage of monomeric dIMP. Hypoxanthine is liberated from DNA directly as a free base. Thus, when poly(dI) x poly(dC) containing both [3H]-dIMP and [32P]dIMP residues was employed as the substrate, 3H-labeled hypoxanthine but no 32P-labeled material was released in ethanol-soluble form. The hypoxanthine-DNA glycosylase presumably acts in DNA repair by preventing deaminated adenine residues from being expressed as mu.

Adenine↗