DNA recognition by the EcoK methyltransferase. The influence of DNA methylation and the cofactor S-adenosyl-L-methionine

The methyltransferase of the EcoK type I restriction/modification system is trimeric, M2S1, where the S subunit determines the sequence specificity of the enzyme. The methyltransferase has a strong preference for hemimethylated substrate DNA and, therefore, we have investigated the effect of the methylation state of DNA on binding by the enzyme, together with the effects on binding of the cofactor S-adenosyl-L-methionine. Our results indicate that the methyltransferase has two non-interacting S-adenosyl-L-methionine binding sites, each with a dissociation constant of 3.60 (+/- 0.42) microM determined by equilibrium dialysis, or 2.21 (+/- 0.29) microM determined by the displacement of a fluorescent probe. Ultraviolet light-induced crosslinking showed that S-adenosyl-L-methionine binds strongly only to the modification (M) subunits. Changes in the sedimentation velocity of the methyltransferase imply a protein conformational change due to S-adenosyl-L-methionine binding. Gel retardation results show that the binding of S-adenosyl-L-methionine to the methyltransferase enhances binding to both specific and non-specific DNAs, but the enhancement is greater for the specific DNA. Differences in binding affinities contribute to the recognition of the specific nucleotide sequence AAC(N)6GTGC by the methyltransferase in preference to a non-specific sequence. In contrast, although the complexes of unmodified and hemimethylated DNAs with the methyltransferase have different mobilities in non-denaturing gels, there appears to be no contribution of binding affinity to the distinction between these two substrates. Therefore, the preference for a hemimethylated substrate must be due to a difference in catalysis.     

Inhibition of DNA methylation by S-adenosylethionine with the production of methyl-deficient DNA in regenerating rat liver

Ethionine, a liver carcinogen, was administered p.o. (300 mg/kg) to rats 17 hr after partial hepatectomy. At 6 hr after administration of the ethionine, hepatic S-adenosylethionine levels were 30- to 40-fold greater than the hepatic level of S-adenosylmethionine. A 10-fold ratio of S-adenosylethionine to S-adenosylmethionine still persited at 24 hr after ethionine administration. When given at 17 hr after partial hepatectomy, ethionine produced a 30% inhibition of DNA synthesis, measured by the incorporation of [methyl-3H]thymidine at 23 to 24 hr after partial hepatectomy (6 to 7 hr after ethionine administration). DNA synthesized during this interval was methyl deficient as judged by the reduced incorporation of radioactivity from L-[methyl-3H]methionine into 5-methylcytosine residues of DNA. In an assay for DNA methylation in vitro using whole nuclei, the methyl-deficient DNA was methylated by S-adenosylmethionine 8 times more than was control DNA; the DNA methylation was competitively inhibited by S-adenosylethionine. These data suggest that S-adenosylethionine, formed in vivo from ethionine, competitively inhibits the methylation of DNA in vivo by S-adenosylmethionine, resulting in the production of methyl-deficient DNA.     

Interference with DNA methyltransferase activity and genome methylation during F9 teratocarcinoma stem cell differentiation induced by polyamine depletion

When ornithine decarboxylase, the initial and highly regulated enzyme in polyamine biosynthesis, is irreversibly inactivated by alpha-difluoromethylornithine, F9 teratocarcinoma stem cells are depleted of putrescine and spermidine and as a result differentiate into a cell type which phenotypically resembles the parietal endoderm cells of the early mouse embryo. Simultaneously the level of decarboxylated S-adenosylmethionine (dcAdoMet), the aminopropyl group donor in spermidine and spermine synthesis, increases dramatically, as the aminopropyl group acceptor molecules (putrescine and spermidine) become limiting. When this excessive accumulation of dcAdoMet is prevented by specific inhibition of the AdoMet decarboxylase activity, the differentiative effect is counteracted, despite the fact that the extent of polyamine depletion remains almost identical. Therefore, it may be concluded that dcAdoMet plays an important role in the induction of differentiation. Moreover, this key metabolite acts as a competitive inhibitor of DNA methyltransferase and is therefore capable of interfering with the maintenance methylation of newly replicated DNA. During the course of F9 cell differentiation, the highly methylated genome is gradually demethylated, and its pattern of gene expression is changed. Our present findings, that the DNA remains highly methylated and that the differentiative process is counteracted when the build-up of dcAdoMet is prevented, provide strong evidence for a causative relation between the level of dcAdoMet and the state of DNA methylation as well as cell differentiation.     

Paramyxovirus phosphoproteins form homotrimers as determined by an epitope dilution assay, via predicted coiled coils

When HA epitope-tagged and untagged Sendai virus (SeV) P proteins are coexpressed and the products reacted with anti-HA, the untagged P protein is also selected because this protein is found as an oligomer. The oligomer was determined to be a homotrimer by coselection studies in which increasing amounts of untagged versus tagged protein were coexpressed, and these findings were extended to mumps virus, a member of the rubulavirus genus. The region of the SeV protein responsible for the oligomerization was localized to residues 344-411. Computer analysis of the 13 Paramyxovirus P proteins in the database revealed that all but one are predicted to form coiled coils in this region, the first of only two regions that can be aligned throughout the entire virus subfamily. The predicted coiled-coil region of the measles virus P protein, when grafted onto the C-terminus of the normally monomeric La protein, led to the efficient oligomerization of this reporter protein. The predicted coiled-coil region of these P proteins thus appears to be sufficient for oligomerization.     

Inhibition of mouse testicular DNA synthesis by mutagens and carcinogens as a potential sample mammalian assay for mutagenesis

In an infant with galactosemia high levels of galactose-1-phosphate in red blood cells and of blood galactose were observed under a "galactose-free' diet. The child did not thrive and developed a liver cirrhosis. At the age of 5 months he died unexpectedly. Post mortem examination revealed in the pancreas and the small intestine changes suggestive of a cystic fibrosis. Since the exogenous administration of galactose by diet could be excluded the endogenous production of significant amounts of galactose-1-phosphate has to be considered.     

Effect of selective cytosine methylation and hydration on the conformations of DNA triple helices containing a TTTT loop structure by FT-IR spectroscopy

5-Methylcytosines have been introduced into triplex-forming-oligonucleotides and shown to extend the pH range over which a triplex forms with a homopurine-homopyrimidine tract of duplex DNA. As a host strand, an oligodeoxypyrimidine with a base sequence of 5'-d(TC)3T4(CT)3 ([CC]) was designed to form a hairpin triplex with a 5'-d-A(GA)2G ([AG6]) purine strand at acidic pH (Tsay, et al., (1995) J. Biomol. Str. Dyn., 13, 1235-1245). We here present results obtained by FT-IR spectroscopy concerning the conformation of the hairpin triplex as a function of the selective substitution of cytosines by 5-methylcytosines in the host strand. Namely, cytosines are substituted by 5-methylcytosines in either the 3'-pyrimidine portion ([CM]) or the 5'-pyrimidine portion ([MC]) or in both ([MM]) of the host strand. The acidic-induced transitions of the equimolar mixtures of the purine target with either of the four pyrimidine oligomers gives rise to different apparent pK values, i.e., [MM].[AG6] (6.2) > [MC].[AG6] (6.0) > [CM].[AG6] (5.7) > [CC].[AG6] (5.2) > single-stranded oligopyrimidines (4.6 +/- 0.2), indicating that cytosine methylation expands the pH range compatible with the hairpin triplex formation regardless of whether the substitution is in the 5'-pyrimidine (Hoogsteen) portion or in the 3'-pyrimidine (Watson-Crick) portion. Thermal denaturation profiles indicated that all the triplexes denatured in a monophasic manner in the pH range of 4.0 to 7.0, and that cytosine methylations in any position of the 16-base pyrimidine oligomer increase the stability of the hairpin triplex DNA. IR spectra recorded in D2O and H2O solutions revealed that cytosine methylation does not significantly influence the conformation of triplex DNA in solution, i.e., all the four triplexes accept a similar sugar conformation, and predominately take on a S-type sugar pucker with a relative proportion of two S-type sugars for one N-type. Furthermore, we also investigated the effect of relative humidity (RH) on the conformation of triplex MC.AG6 in hydrated films, and found that the conformational change induced by the decrease of RH, from predominant S-type to primary N-type sugar pucker, might first occur in the purine strand at 86% RH.     

Structure and expression of the human SM22alpha gene, assignment of the gene to chromosome 11, and repression of the promoter activity by cytosine DNA methylation

The pore-forming alpha 1 subunit of L-type calcium (Ca2+) channels is the molecular target of Ca2+ channel blockers such as phenylalkylamines (PAAs). Association and dissociation rates of (-)devapamil were compared for a highly PAA-sensitive L-type Ca2+ channel chimera (Lh) and various class A Ca2+ channel mutants. These mutants carry the high-affinity determinants of the PAA receptor site in a class A sequence environment. Apparent drug association and dissociation rate constants were significantly affected by the sequence environment (class A or L-type) of the PAA receptor site. Single point mutations affecting the high-affinity determinants in segments IVS6 of the PAA receptor site, introduced into a class A environment, reduced the apparent drug association rates. Mutation I1811M in transmembrane segment IVS6 (mutant AL25/-I) had the highest impact and decreased the apparent association rate for (-)devapamil by approximately 30-fold, suggesting that this pore-lining isoleucine in transmembrane segment IVS6 plays a key role in the formation of the PAA receptor site. In contrast, apparent drug dissociation rates of Ca2+ channels in the resting state were almost unaffected by point mutations of the PAA receptor site.     

Testing the feasibility of DNA typing for human identification by PCR and an oligonucleotide ligation assay

The use of DNA typing in human genome analysis is increasing and finding widespread application in the area of forensic and paternity testing. In this report, we explore the feasibility of typing single nucleotide polymorphisms (SNPs) by using a semiautomated method for analyzing human DNA samples. In this approach, PCR is used to amplify segments of human DNA containing a common SNP. Allelic nucleotides in the amplified product are then typed by a colorimetric implementation of the oligonucleotide ligation assay (OLA). The results of the combined assay, PCR/OLA, are read directly by a spectrophotometer; the absorbances are compiled; and the genotypes are automatically determined. A panel of 20 markers has been developed for DNA typing and has been tested using a sample panel from the CEPH pedigrees (CEPH parents). The results of this typing, as well as the potential to apply this method to larger populations, are discussed.     

DNA methylation and the promotion of flowering by vernalization

We have tested the hypothesis that the promotion of flowering by prolonged exposure to low temperatures (vernalization) is mediated by DNA demethylation [Burn, J. E., Bagnall, D. J., Metzger, J. M., Dennis, E. S. & Peacock, W. J. (1993) Proc. Natl. Acad. Sci. USA 90, 287-291]. Arabidopsis plants that have reduced levels of DNA methylation because of the presence of a methyltransferase (METI) antisense gene flowered earlier than untransformed control plants, without the need for a cold treatment. Decreased DNA methylation mutants (ddm1) also flowered earlier than the wild-type progenitor under conditions where they respond to vernalization. We conclude that demethylation of DNA is sufficient to cause early flowering, and we have found that the promotion of flowering is directly proportional to the decrease in methylation in METI antisense lines. The early-flowering phenotype was inherited in sexual progeny, even when the antisense transgene had been lost by segregation. Methyltransferase antisense plants with low DNA methylation levels responded to a low-temperature treatment by flowering even earlier than their untreated siblings indicating that the promotion of flowering by cold and by demethylation was additive when neither treatment saturated the early-flowering response. As in untransformed control plants, the cold-induced early-flowering signal was reset in progeny of METI antisense plants. These observations suggest that the demethylation brought about by a METI antisense can account for some properties of vernalization, but not for the need for revernalization in each generation.     

Guanine tetraplex formation by short DNA fragments containing runs of guanine and cytosine

Using CD spectroscopy, guanine tetraplex formation was studied with short DNA fragments in which cytosine residues were systematically added to runs of guanine either at the 5' or 3' ends. Potassium cations induced the G-tetraplex more easily with fragments having the guanine run at the 5' end, which is just an opposite tendency to what was reported for (G+T) oligonucleotides. However, the present (G+C) fragments simultaneously adopted other conformers that complicated the analysis. We demonstrate that repeated freezing/thawing, performed at low ionic strength, is a suitable method to exclusively stabilize the tetraplex in the (G+C) DNA fragments. In contrast to KCl, the repeated freeze/thaw cycles better stabilized the tetraplex with fragments having the guanine run on the 3' end. The tendency of guanine blocks to generate the tetraplex destabilized the d(G5).d(C5) duplex whose strands dissociated, giving rise to a stable tetraplex of (dG5) and single-stranded (dC5). In contrast to d(G3C3) and d(G5C5), repeated freezing/thawing induced the tetraplex even with the self-complementary d(C3G3) or d(C5G5); hence the latter oligonucleotides preferred the tetraplex to the apparently very stable duplex. The tetraplexes only included guanine blocks while the 5' end cytosines interfered neither with the tetraplex formation nor the tetraplex structure.     

Concentration differences between serum and plasma of the elements cobalt, iron, mercury, rubidium, selenium and zinc determined by neutron activation analysis

The differences in concentrations of cesium, cobalt, iron, mercury, rubidium, selenium and zinc between serum and plasma were examined with the aid of instrumental neutron activation analysis. Eighty serum and plasma samples obtained from 13 donors were compared. Serum was prepared in plastic tubes immediately after clotting, and plasma was separated with heparin as anticoagulant. No significant differences in the concentrations of cesium, cobalt, mercury and selenium were observed. However, the concentrations of iron, rubidium and zinc were significantly higher in serum than in plasma. The average differences were 322, 12 and 20 ng/ml for iron rubidium and zinc, respectively. The average differences found for cesium, rubidium and zinc were far below that which can be expected from a complete, or considerable release of these elements from platelets which aggregate or disintegrate during the clotting process in preparing serum.     

Incision at DNA G.T mispairs by extracts of mammalian cells occurs preferentially at cytosine methylation sites and is not targeted by a separate G.T binding reaction

We have investigated the specificities of G.T mismatch binding proteins and of G.T mismatch cleavage in extracts of mammalian cells. G.T mismatch-specific protein:DNA complex formation by cell extracts was independent of the local sequence context of the mismatch. Cell extracts performed similar levels of protein binding to DNA substrates in which a single G.T mispair was preceded by T, G, A, C, or 5-meC. In contrast, incision by extracts of the T-containing strand of a G.T mismatch exhibited a strong sequence specificity and efficient strand cleavage was only observed when the mismatched G was in a CpG sequence. Thus, oligonucleotides containing either CpgGpT or 5meCpGGpT were efficiently incised, but not those containing GpGCpT, ApGTpT, or TpGApT sequences. Cell lines made resistant to the alkylating agent N-methyl-N-nitrosourea have previously been found to be defective in a G.T mismatch binding reaction. The defect in binding by extracts prepared from these cells extended to G.T mismatches in several sequence contexts. The variant extracts nevertheless incised G.T mismatches normally suggesting that this particular binding activity is not required for incision. The data indicate that incision by this activity is targeted to the CpG sequences in which G.T mismatches are formed by the mutagenic deamination of DNA 5-methylcytosine. In this regard the repair pathway resembles the very short patch (vsp) repair pathway in Escherichia coli.     

Inhibition of human angiogenin by DNA aptamers: nuclear colocalization of an angiogenin-inhibitor complex

Specific ligands (aptamers) for angiogenin were selected from a 72-mer oligodeoxynucleotide library consisting of 28 randomized positions flanked by two constant regions of 22 residues each. From a starting pool of approximately 10(14) molecules, 19 angiogenin-binding ligands were obtained. Among them, two oligonucleotides showed significant inhibition of the ribonucleolytic activity of angiogenin with apparent Kis of 0.65 and 0.60 micro M, respectively. One of them was shortened on the basis of its secondary structure to provide a 45-mer oligonucleotide that retained much of the inhibitory properties of the parent molecule. It inhibits both the angiogenic and cell proliferative activities of angiogenin but does not interfere with its nuclear translocation in human endothelial cells. Importantly, the inhibitor is cotranslocated to the nucleus with angiogenin in a approximately 1:1 stoichiometric ratio. These results demonstrate that the inhibition of angiogenin-induced cell proliferation and angiogenesis by the oligonucleotide is due to suppression of the ribonucleolytic activity of angiogenin, an event that occurs most likely within the cell nucleus.     

DNA cleavage, antiviral and cytotoxic reactions photosensitized by simple enediyne compounds

Very potent antibiotic antitumor natural products contain a enediyne moiety which, upon thermal activation, is capable of abstracting hydrogens from DNA. 1,6-Diphenyl-3-hexene-1,5-diyne was selected as a candidate for inducing DNA strand breaks photochemically. Easily interconverted with light, both geometric isomers 1 and 2 were expected to be phototoxic. As anticipated, they photosensitized the production of strand breaks in double-stranded supercoiled pBR322, and in single-stranded M13 DNA. The DNA cleavage reactions were favored by the presence of oxygen and were inhibited by ethanol. Preliminary experiments with the (Z)-isomer indicated moderate light-dependent antiviral activity against human immunodeficiency virus (HIV), Sindbis virus, and mouse cytomegalovirus. The enediynes were cytotoxic to Escherichia coli, a gram-negative organism, to Streptococcus faecalis, a gram-positive organism, to Daphnia magna and to fish (Pimephales promelas), but only in the presence of light. The production of o-terphenyl, the expected product of Bergman cyclization of 1, could not be confirmed. However, both 1 and 2 photosensitized the formation of singlet oxygen and of superoxide anion radical, and photodynamic reactions could have been responsible for some of the phototoxic reactions observed.     

Are somatic cells inherently deficient in methylation metabolism? A proposed mechanism for DNA methylation loss, senescence and aging

A mechanism of aging is proposed for mammals and other vertebrates. In this mechanism, most somatic cells have inherent deficiencies in methylation metabolism with respect to their capacity to methylate DNA. This leads to incomplete DNA methylation in each cell cycle which, accumulated over many cell cycles, contributes to genetic instability, senescence and cancer. These proposed metabolic deficiencies are present from the time somatic cells are young, yet it is only after many cell divisions that deleterious effects are realized. In nature, most animals have reproduced or have been killed by predators or other environmental hazards before they can be greatly affected by these deficiencies. These deficiencies evolved in animals eating a balance of nutrients from nature. Evidence from the literature is reviewed which establishes that methylation is lost from the DNA of many mammalian somatic cells as they age both in vivo and in vitro, and that DNA methylation levels are influenced by factors, such as diet, that affect methylation metabolism. Partially correcting the proposed deficiencies is considered as a possible molecular mechanism by which caloric restriction extends lifespan. Other possible dietary and transgenic means to correct the proposed deficiencies and extend lifespan are discussed.     

Activities of thyroid hormones and related compounds in an in vitro thymocyte assay

Prior studies have demonstrated that the thyroid hormones L-triiodothyronine and L-thyroxine stimulate the rapid uptake of 1-amino-cyclopentane-1-carboxylic acid into isolated rat thymocytes. In the present study the effects of several groups of thyroid hormones and structurally related compounds were investigated to determine the structure-function relations required for stimulation of this membrane process. Particular attention was given to (a) analogues with modifications at the oxygen bridge, the phenolic hydroxyl group, and the group at the 3' position of the outer ring, and (b) the steric orientation of the thyroid hormones. The following were found to be important for maximal activity: (a) the L-isomer configuration, (b) the presence of a 4'-hydroxyl group, (c) the presence of one substituent in both the inner and outer rings (3 and 3' positions), (d) the distal orientation of the 3' substituent in the outer ring of L-triiodothyronine, and (e) the lipophilic character of the 3' substituent. Of lesser importance was the presence of halogen atoms, or an oxygen atom in the ether position. Since these structure-function relations seen in thymocytes parallel in many respects those relations seen in whole-animal studies, it is believed that thymocytes will be a useful tool for further studies of thyroid hormones and their analogues.