Use of a dodecadeoxynucleotide to study repair of the O4-methylthymine lesion

A dodecadeoxynucleotide of defined sequence containing O4-methylthymine was labeled at the 5' end with [32P] by the reaction with (gamma-32P]ATP and polynucleotide kinase. Extracts prepared from bacterial and mammalian sources such as the human cell lines, HeLa and HT29, and rat liver were incubated with the labeled, methylated dodecamer to determine the extent of repair of the lesion. The labeled, demethylated dodecamer was separated from the labeled methylated dodecamer on a reverse-phase column using a shallow methanol gradient. There was complete repair of O4-methylthymine by the E. coli alkyltransferase upon incubation for 4 h at 37 degrees C. There was no detectable amount of demethylated product formed upon incubation with HeLa or HT29 cell extract for the same incubation period. There was also no repair of the O4-methylthymine lesion in the presence of crude rat-liver extract. However, the rat-liver extract alone degraded the methylated substrate completely, and the assay had to be conducted in the presence of NaF, AMP and unlabeled, nonmethylated dodecamer to prevent this. The results obtained from this assay, which is at least an order of magnitude more sensitive than previous methods, are in agreement with previous results that the mammalian alkyltransferase is specific for O6-alkylguanine repair.     

DNA-synthesis with methylated poly(dA-dT) templates: possible role of O4-methylthymine as a pro-mutagenic base.

The alternating copolymer poly(dA-dT) has been methylated with either dimethyl sulphate (DMS) or N-methyl-N-nitrosourea (MNU) and the levels of the various methylation products determined. In addition to the methylated adenines formed by both methylating agents, MNU resulted also in the formation of 3-methylthymine, O4-methylthymine and phosphotriesters. The methylated polymers have been ution of complementary and non-complementary nucleotides determined. With the DMS methylated template no wrong nucleotide incorporation was detectable, but with the MNU methylated polymer the incorporation of dGMP was observed. The amount of dGMP incorporated correlated with the level of O4-methylthymine in the template over the range of methylation studied. The results indicate that O4-methylthymine is capable of miscoding on a one-to-one basis while the products of DMS methylation (1-, 3- and 7-methyladenines), and also possibly the phosphotriesters, do not lead to any misincorporation.     

Identification of mammalian aspartate-4-decarboxylase

Several animal tissues were examined for aspartate-4-decarboxylase (EC 4.1.1.12) activity. Highest activity was seen in murine livers, in rodent livers, and in rodent kidneys. The rat liver enzyme was membrane associated and could be solubilized and partially purified with the aid of detergents. The purification studies, and studies on the stoichiometry and kinetics of the reaction, showed that aspartate is directly converted to alanine. Such a metabolic reaction had not been reported before in animals. The rat liver enzyme differed significantly from the microbial aspartate-4-decarboxylases. Among other things, the rat liver beta-decarboxylase could be purified away from a cysteine sulfinate desulfinase activity. Also, unlike the bacterial enzymes, the mammalian beta-decarboxylase could not be inactivated by preincubation with aspartate or cysteine sulfinate. These later observations strongly suggest that the mammalian aspartate-4-decarboxylase does not have an inherent transaminase activity. Like many decarboxylases, rat liver aspartate-4-decarboxylase could be inhibited by reagents which react with carbonyl groups; however, the enzyme showed no dependence on pyridoxal 5'-phosphate.     

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.     

Receptor-Mediated Transfer of DNA–Galactosylated Poly-L-lysine Complexes into Mammalian Cells in vitro and in vivo

With the goal of developing non-viral techniques for exogenous gene delivery into mammalian cells, we have studied receptor-mediated gene transfer using complexes of plasmid DNA and galactosylated poly-L-lysine, poly(L-Lys)Gal. To evaluate the optimal parameters for efficient gene transfer into human hepatoma HepG2 cells by the DNA–poly(L-Lys)Gal complexes, the bacterial reporter genes lacZ and cat were used. Examination of the reporter gene expression level showed that the efficiency of DNA delivery into the cells depends on the structure of DNA–poly(L-Lys)Gal complexes formed at various ionic strength values. The efficiency of DNA transfer into the cells also depends on DNA/poly(L-Lys)Gal molar ratio in the complexes. Plasmid vector carrying human apolipoprotein A-I (apoA-I) gene was injected as its complex with poly(L-Lys)Gal into rat tail vein. Some level of ApoA-I was detected in the serum of the injected rats. Also, the human apoA-I-containing plasmid was found to be captured specifically by the rat liver cells and transported into the cell nuclei, where it can persist as an episome-like structure for at least a week. After repeated injections of DNA–poly(L-Lys)Gal complexes, the level of human ApoA-I in rat serum increases, probably, due to accumulation of functional human apoA-I gene in the liver cell nuclei. The data seem to be useful for the development of non-viral approaches to gene therapy of cardiovascular diseases.     

Repair of DNA O-alkylation damage by various human organs

Protein extracts from human adult liver, fetal liver, intestine, brain, kidney, lung and skin were tested against poly(dT)methylated X poly(dA), poly(dA)methylated X poly(dT) and methylated DNA. The suitability of various substrates was established in assays using E. coli extracts that removed O4-methylthymidine (O4-MedT), O2-MedT, and O6-methylguanine (O6-MeG). The human extracts efficiently removed O6-MeG and N3-methyladenine from methylated substrates. The adult liver exhibited low and fetal tissues negligible removal of O4-MedT. Only the liver showed limited removal of O2-MedT. The poor removal of the miscoding base O4-MedT by human organs could be an important factor in carcinogen induced mutagenesis, carcinogenesis and teratogenesis.     

Biochemical characterization of rat P450 2C11 fused to rat or bacterial NADPH-P450 reductase domains

cDNAs coding for rat P450 2C11 fused to either a bacterial (the NADPH-cytochrome P450 BM3 reductase domain of P450 BM3) or a truncated form of rat NADPH-P450 reductases were expressed in Escherichia coli and characterized enzymatically. Measurements of NADPH cytochrome c reductase activity showed fusion-dependent increases in the rates of cytochrome c reduction by the bacterial or the mammalian flavoprotein (21 and 48%, respectively, of the rates observed with nonfused enzymes). Neither the bacterial flavoprotein nor the truncated rat reductase supported arachidonic acid metabolism by P450 2C11. In contrast, fusion of P450 2C11 to either reductase yielded proteins that metabolized arachidonic acid to products similar to those obtained with reconstituted systems containing P450 2C11 and native rat P450 reductase. Addition of a 10-fold molar excess of rat P450 reductase markedly increased the rates of metabolism by both fused and nonfused P450s 2C11. These increases occurred with preservation of the regioselectivity of arachidonic acid metabolism. The fusion-independent reduction of P450 2C11 by bacterial P450 BM3 reductase was shown by measurements of NADPH-dependent H(2)O(2) formation [73 +/- 10 and 10 +/- 1 nmol of H(2)O(2) formed min(-)(1) (nmol of P450)(-)(1) for the reconstituted and fused protein systems, respectively]. These studies demonstrate that (a) a self-sufficient, catalytically active arachidonate epoxygenase can be constructed by fusing P450 2C11 to mammalian or bacterial P450 reductases and (b) the P450 BM3 reductase interacts efficiently with mammalian P450 2C11 and catalyzes the reduction of the heme iron. However, fusion is required for metabolism and product formation.     

Subunit composition of rat liver glutathione S-transferases

The plasmid pGTR112 contains partial coding sequences for one of the rat liver glutathione S-transferase subunits. We have used immobilized pGTR112 DNA to select for complementary and homologous liver poly(A)-RNAs under conditions of increasing stringency for hybridization. Each fraction of selected poly(A)-RNAs was assayed by in vitro translation followed by immunoprecipitation. A total of four distinct polypeptides precipitated by antiserum against rat liver glutathione S-transferases were resolved by NaDodSO₄ polyacrylamide gel electrophoresis. They are separated into two pairs according to the sequence homology of their poly(A)-RNAs with the pGTR112 DNA. Purified rat liver glutathione S-transferases can be resolved on gradient NaDodSO₄ polyacrylamide gels into four polypeptides. There should be ten isozymes of different binary combinations from four distinct subunits for the rat liver glutathione S-transferases.     

Immunological comparison of the proteins of chicken and rat liver ribosomes.

A comparison of the proteins of chicken and rat liver ribosomes using immunochemical techniques was undertaken. The procedures included quantitative precipitation, passive hemagglutination, and immunodiffusion on Ouchterlony plates. The results indicate that antisera specific for chicken or rat liver ribosomes recognize only about 20% of common determinants. While there are important reservations, the results suggest extensive differences in the proteins of rat and chicken liver ribosomes. Despite those differences, rat and chicken liver ribosomal proteins maintain some homologous sequences present in bacterial ribosomal proteins. An enriched antibody preparation against chicken 80 S ribosomes inhibited the poly(U)-directed synthesis of polyphenylalanine and the elongation factor G (EF-G)-catalyzed binding of [3H]GDP to Escherichia coli ribosomes. Thus, chicken liver ribosomes, like ribosomes from rat liver and yeast, must have proteins homologous with those of E. coli ribosomes.     

Expression of the truncated E. coli O6-methylguanine methyltransferase gene in repair-deficient human cells and restoration of cellular resistance to alkylating agents

We have constructed a truncated E. coli O6-methylguanine methyltransferase (MT) gene (ada gene) to express the MT activity for O6-methylguanine and O4-methylthymine but not for methylphosphotriester in human cells and transferred it into Mer- HeLa MR cells. The transfectant cells expressed the truncated E. coli MT were resistant to alkylating agents as same as the transfectant cells with the intact ada gene in cell killing, sister-chromatid exchange induction and host-cell reactivation of adenovirus 5. These results strongly suggest that methylphosphotriester may not contribute to the biological effect of alkylating agents in human cells.     

Peptidyl transferase centres of rat and yeast ribosomes. Different response to modification of protein amino groups

Modification of rat liver ribosomes with dimethylmaleic anhydride, a reagent for protein amino groups, causes a large stimulation of peptidyl transferase activity assayed by the "fragment" reaction, as well as the inactivation of poly(U)-directed polyphenylalanine synthesis. In contrast to rat ribosomes, the peptidyl transferase of yeast ribosomes is little affected by modification. Although other interpretations are not excluded, these results might be due to differences between the peptidyl transferase centres of mammalian and yeast ribosomes.     

The suicidal DNA repalr methyltransferases of microbes

Virtually every organism so far tested has been found to possess an extremely efficient DNA repair mechanism to ensure that certain alkylated oxygens do not accumulate in the genome. The repair is executed by DNA methyltransferases (MTases) which repair DNA O6-methylguanine (O6MeG), O4-methylthymine (O4MeT) and methylphosphotriesters (MePT). The mechanism is rather extravagant because an entire protein molecule is expended for the repair of just one, or sometimes two, O-alkyl DNA adduct(s). Cells profit from such an expensive transaction by earning protection against death and mutation by alkylating agents. This review considers the structure, function and biological roles of a number of well-characterized microbial DNA repair MTases.     

Conformational properties of the osmium tetraoxide bispyridine ester of 1-methylthymine and a comment on the linearity of the trans O=Os=O group.

The preparation and crystal and molecular structure of the osmium tetraoxide bispyridine ester of 1-methylthymine are reported. The complex crystallizes in the triclinic system, space group P1, with a = 11.493(6)A, b = 16.655(7)A, c = 6.082(2)A, alpha = 92.07(3) degrees, beta = 90.58(3) degrees, gamma = 71.36(4) degrees, V = 1102.4 A3, Dm = 1.85(1) g cm-3, DC = 1.84 g cm-3. The unit cell contains 2 osmium tetraoxide bispyridine esters of 1-methylthymine, 2 waters of crystallization and 1 disordered pyridine of solvation. Intensities for 3814 independent reflections were collected by counter methods. The structure was solved by standard heavy-atom techniques and has been refined by full-matrix least squares, based on F, to a final R value of 0.065. The osmium complex binds as a cis osmate ester to the C(5)-C(6) bond of the methylated pyrimidine in a fashion which is expected to be similar to the binding of the complex to thymidine residues in nucleic acids. The conformation of the 1-methylthymine ester is that of a half chair with C(6) showing a substantial deviation, 0.55 A, from the best mean plane of the thymine moiety. The primary coordination sphere about the Os(VI) atom is completed by 2 axial Os=O bonds and the binding of the 2 pyridine ligands in cis positions in the equatorial plane containing the ester linkages. The O=Os=O group is substantially nonlinear, 164.0(5) degrees, and this nonlinearity is attributed to intracomplex electronic effects.     

Heme degradation as catalyzed by a recombinant bacterial heme oxygenase (Hmu O) from Corynebacterium diphtheriae.

Hmu O, a heme degradation enzyme in the pathogen Corynebacterium diphtheriae, catalyzes the oxygen-dependent conversion of hemin to biliverdin, carbon monoxide, and free iron. A bacterial expression system using a synthetic gene coding for the 215-amino acid, full-length Hmu O has been constructed. Expressed at very high levels in Escherichia coli BL21, the enzyme binds hemin stoichiometrically to form a hexacoordinate high spin hemin-Hmu O complex. When ascorbic acid is used as the electron donor, Hmu O converts hemin to biliverdin with alpha-hydroxyhemin and verdoheme as intermediates. The overall conversion rate to biliverdin is approximately 4-fold slower than that by rat heme oxygenase (HO) isoform 1. Reaction of the hemin-Hmu O complex with hydrogen peroxide yields a verdoheme species, the recovery of which is much less compared with rat HO-1. Reaction of the hemin complex with meta-chloroperbenzoic acid generates a ferryl oxo species. Thus, the catalytic intermediate species and the nature of the active form in the first oxygenation step of Hmu O appear to be similar to those of the mammalian HO. However, the considerably slow catalytic rate and low level of verdoheme recovery in the hydrogen peroxide reaction suggest that the active-site structure of Hmu O is different from that of its mammalian counterpart.     

Methylated purines formed in DNA by dimethylnitrosamine in rats previously exposed to hepatotoxic and hepatocarcinogenic regimes: effects on the repair of O6-methylguanine

Studies of mammalian systems for the repair of O6-methylguanine in DNA have revealed large differences in the capacities of tissues and cells to perform this function and in the case of rat liver it has been shown that the O6-methylguanine repair system can be stimulated by exposure to hepatotoxic and hepatocarcinogenic regimes. In this report an assessment is made of possible relationships between toxic liver injury, DNA synthesis, cell proliferation and DNA repair by treating Wistar rats with agents selected to provide differing degrees of liver involvement. The effects of long-term (20 week) treatments with acetylaminofluorene (15 mg/kg/day), quinoxaline 1,4-dioxide (10 mg/kg/day), 4-aminobiphenyl-HCl (15 mg/kg/day) and pronethalol (20 mg/kg/day) were assessed, using the same strain of animals in which the original toxicity and carcinogenicity data were obtained. Repair of O6-methylguanine produced in liver DNA by a low, non-toxic dose (2 mg/kg) of [14C]dimethylnitrosamine was increased 3-4-fold throughout the period of treatment with acetylaminofluorene, to a lesser extent by quinoxaline 1,4-dioxide and 4-aminophenyl-HCl and not at all in the case of pronethalol. No evidence was obtained to indicate a direct relationship between O6-methylguanine repair and either the induced hepatotoxicity or the ensuing increased rates of DNA synthesis which occur following exposure to these agents.     

A simple method for the solid phase synthesis of oligodeoxynucleotides containing O4-alkylthymine.

A route to prepare the cyanoethyl-phosphoramidite monomer of O4-alkylthymine and a method for the routine solid-phase synthesis of oligodeoxynucleotides containing O4-alkylthymine are described. This method has been used to make DNA sequences up to 48 bases in length. The amino function of the adenine and guanine in the sequence were protected with the phenoxyacetyl group, and that of cytosine with the isobutyryl group. The phosphodiesters were protected with the cyanoethyl group. This allowed complete deprotection of the oligomer with alkoxide ions (methanol/1,8- diazabicyclo[5.4.0]undec-7-ene (DBU) for the oligomers containing O4-methylthymine, or ethanol/DBU for those containing O4-ethylthymine) thus avoiding the use of ammonia which is known to attack the O4-alkylthymine to form 5-methylcytosine. There was no detectable loss of the alkyl group to form thymine.     

Regulation of urea synthesis in sublobular regions of the liver lobule by oxygen

Periportal and pericentral regions of the liver lobule were isolated from perfused rat liver using a micropunch and incubated in Krebs-Henseleit buffer (pH 7.6) containing 2% poly(ethylene glycol) in Eagle's basal medium, PMSF (50 micrograms/ml) and leupeptin (20 micrograms/ml) for 2 h at 25 degrees C under and O2/CO2 (95:5%) gas phase. Maximal rates of urea production from ammonium chloride were 96.4 +/- 8.7 and 32.8 +/- 5.4 mumol/g per h at 800 and 200 microM O2. Thus, urea synthesis was 2-3-times greater at high than low O2 tension in plugs from periportal and pericentral regions of the liver lobule.     

Measurement of the complexity and diversity of poly(adenylic acid) containing messenger RNA from rat liver.

The complexity of rat liver poly (A)+ messenger RNA (mRNA) has been measured by analysis of the kinetics of hydridization with both complementary DNA (cDNA) and single copy DNA. The complementary DNA-poly(A)+ mRNA hybridization reaction demonstrates the existence of three abundance classes representing 18, 37, and 45% of the cDNA and 4, 290, and 24 000 different 1800-nucleotide sequences respectively. The poly(A)+ mRNA driven single copy DNA hybridization reaction reveals a single major transition accounting for 1.9% of the haploid rat genome. The kinetics of the poly(A)+ mRNA driven single copy DNA reaction suggest that approximately 45% of the mass of the mRNA population contains over 95% of the complexity. Although higher than previous estimates, the base sequence complexities of rat liver poly(A)+ mRNA measured in these two ways are in good agreement, suggesting that the technique of poly(A)+ mRNA-cDNA hybridization may be used in approximating the complexity as well as abundance of a messenger RNA population. DNA-driven cDNA reactions reveal that about 10% of rat liver poly(A)+ mRNA is transcribed from repetitive sequences in the rat genome.     

Separation of the SOS-dependent and SOS-independent components of alkylating-agent mutagenesis.

Escherichia coli plasmids containing the rpsL+ gene (Strs phenotype) as the target for mutation were treated in vitro with N-methyl-N-nitrosourea. Following fixation of mutations in E. coli MM294A cells (recA+ Strs), an unselected population of mutant and wild-type plasmids was isolated and transferred into a second host, E. coli 6451 (recA Strr). Strains carrying plasmid-encoded forward mutations were then selected as Strr isolates, while rpsL+ plasmids conferred the dominant Strs phenotype in the second host. Mutation induction and reduced survival of N-methyl-N-nitrosourea-treated plasmids were shown to be dose dependent. Because this system permitted analysis and manipulation of the levels of certain methylated bases produced in vitro by N-methyl-N-nitrosourea, it afforded the opportunity to assess directly the relative roles of these bases and of SOS functions in mutagenesis. The methylated plasmid DNA gave a mutation frequency of 6 X 10(-5) (a 40-fold increase over background) in physiologically normal cells. When the same methylated plasmid was repaired in vitro by using purified O6-methylguanine DNA methyltransferase (to correct O6-methylguanine and O4-methylthymine), no mutations were detected above background levels. In contrast, when the methylated plasmid DNA was introduced into host cells induced by UV light for the SOS functions, rpsL mutagenesis was enhanced eightfold over the level seen without SOS induction. This enhancement of mutagenesis by SOS was unaffected by prior treatment of the DNA with O6-methylguanine DNA methyltransferase. These results demonstrate a predominant mutagenic role for alkylation lesions other than O6-methylguanine or O4-methylthymine when SOS functions are induced. The mutation spectrum of N-methyl-N-nitrosourea under conditions of induced SOS functions revealed a majority of mutagenic events at A . T base pairs.