Inactivation of de novo DNA methyltransferase activity by high concentrations of double-stranded DNA

The activity of eukaryotic DNA methyltransferase diminishes with time when the enzyme is incubated with high concentrations (200–300 μg/ml) of unmethylated double-stranded Micrococcus luteus DNA. Under similar conditions, single-stranded DNA induces only a limited decrease of enzyme activity. The inactivation process is apparently due to a slowly progressive interaction of the enzyme with double-stranded DNA that is independent of the presence of S-adenosyl-l-methionine. The inhibited enzyme cannot be reactivated either by high salt dissociation of the DNA-enzyme complex or by extensive digestion of the DNA. Among synthetic polydeoxyribonucleotides both poly(dG-dC) · poly(dG-dC) and poly(dA-dT) · poly(dA-dT), but not poly(dI-dC) · poly(dI-dC), cause inactivation of DNA methyltransferase. This inactivation process may be of interest in regulating the ‘de novo’ activity of the enzyme.     

Preferential binding of DNA methyltransferase and increased de novo methylation of deoxyinosine containing DNA

Mammalian DNA-cytosine 5-methyltransferases methylate cytosines in deoxyinosine containing DNA polymers more rapidly than in other synthetic or naturally occurring DNAs. The initial methylation rate of poly(dI-dC) X poly(dI-dC) is about 10-times higher than that of poly-(dG-dC) X poly(dG-dC) or of the native Micrococcus luteus DNA. In competitive binding experiments, DNA methyltransferase has about 10-fold higher affinity for the dI-containing alternating DNA polymer than for poly(dG-dC) X poly(dG-dC). The observed high methyl accepting capacity of poly(dI-dC) X poly(dI-dC) may be a useful methodological advance to determine de novo DNA methyltransferase activity in extracts of mammalian cells.     

Isolation and characterization of proteins that stimulate the activity of mammalian DNA methyltransferase

Previously, the purification of DNA methyltransferase from murine P815 mastocytoma cells by immunoaffinity chromatography was described (Pfeifer, G.P., Grünwald, S., Palitti, F., Kaul, S., Boehm, T.L.J., Hirth, H.P. and Drahovsky, D. (1985) J. Biol. Chem. 260, 13787-13793). Proteins that stimulate the enzymatic activity of DNA methyltransferase have been purified from the same cells. These proteins, which partially coelute with DNA methyltransferase from DEAE-cellulose and heparin-agarose, are separated from the enzyme during the immunoaffinity purification step. A further purification of the stimulating proteins was achieved by butanol extraction, DEAE-cellulose chromatography and gel filtration on Superose 12. Two DNA methyltransferase-stimulating protein fractions were obtained. SDS-polyacrylamide gel electrophoresis of one fraction showed a single polypeptide with a molecular mass of 29 kDa. The second fraction consisted of 5 or 6 polypeptides with molecular masses 78-82 and 51-54 kDa. The proteins stimulate both de novo and maintenance activity of DNA methyltransferase about 3-fold. They enhance the methylation of any natural DNA and of poly[(dI-dC).(dI-dC)] but inhibit the methylation of poly[(dG-dC).(dG-dC)]. The purified proteins do not form a tight complex with DNA methyltransferase; however, they bind both to double-stranded and single-stranded DNA. The sequence specificity of DNA methyltransferase is obviously altered in presence of these proteins.     

Mode of reversible binding of neocarzinostatin chromophore to DNA: base sequence dependency of binding.

The reversible binding of neocarzinostatin chromophore to polynucleotides was studied in order to understand the molecular basis of its base sequence-specificity in DNA damage production. Studies of the spectroscopic and thermodynamic properties of chromophore-polynucleotide interactions reveal that the binding of the chromophore to poly(dA-dT) is qualitatively and quantitatively different from that to poly(dG-dC) (and poly(dI-dC]. From these and other experiments using double-stranded mixtures of homopolynucleotides, it is proposed that the observed A T specific intercalation might result from differential recognition of minor variations in the B-DNA type structure by the chromophore.     

Quantitative analysis of DNA-porphyrin interactions

The binding of manganese(III)-tetra(4-N-methylpyridyl)porphyrin (MnTMpyP) with synthetic poly(dA-dT)2, poly(dI-dC)2, and poly(dG-dC)2 DNAs as well as calf thymus (CT) DNA has been quantitatively studied in detail using induced CD (circular dichroism) spectroscopy in the Soret absorption band. The CD spectra, which changed greatly depending on the porphyrin to DNA base-pair molar ratio (r), were normalized with respect to DNA concentration and deconvoluted. Three independent component binding modes (named mode 1, 2, and 3 in the order of increasing r values) were identified, which successfully simulated the observed CD spectra with negligibly small residuals for a wide range of r values. In the case of poly(dA-dT)2, poly (dI-dC)2, and CT DNA, all the three modes appeared, whereas in the case of poly(dG-dC)2 DNA, only modes 1 and 3 appeared in the r range studied. The r dependence of each binding mode, i.e., its relative affinity toward DNA, has been revealed by this analysis. Mode 1, which appeared as a single binding mode at very low r values (r < or = ca. 0.05), was inhibited by the addition of methyl green, a drug that preferentially binds to the major groove of poly (dA-dT)2 DNA. Berenil, a known minor groove binder to poly(dA-dT)2 or poly(dI-dC)2 DNA, inhibited modes 2 and 3. From these inhibition experiments as well as comparison of the component spectra for DNAs of different sequence, a binding site on DNA was proposed for each component binding mode. The number of DNA base pairs covered by a single molecule of porphyrin was estimated.     

Purification and properties of a novel DNA methyltransferase from cultured rice cells

DNA methyltransferase activity has been observed in a total crude homogenate of rice cells grown in suspension culture using either native plant DNA or, under the conditions used, the more responsive hemimethylated poly (dI-MedC).poly(dI-dC). Using the latter substrate we have purified an enzyme fraction 380-fold by salt extraction of chromatin, DEAE cellulose and phosphocellulose. This purified fraction showed enzyme activity only with poly (dI-MedC).poly(dI-dC) thus suggesting the occurrence in plants of a DNA methyltransferase specific for hemimethylated DNA. A Mr value of 54000 was calculated on the basis of the sedimentation coefficient which was determined by sucrose density gradient centrifugation. Apparent Km values for poly (dI-MedC).poly(dI-dC) and S-adenosyl-L-methionine were found to be 17 micrograms/ml and 2.6 microM, respectively.     

Vibrational circular dichroism studies of the A-to-B conformational transition in DNA.

The vibrational circular dichroism (VCD) spectra of several natural DNAs as well as tRNA, poly(dG-dC).poly(dG-dC), and poly(dA-dT).poly(dA-dT) are reported for the base deformation modes in the IR region from 1700 to 1550 cm-1 for the polymers in D2O as well as in high alcohol dehydrating conditions. Spectra of both the B- and A-forms were identified. The A-form DNA VCD, not previously reported, has characteristics that can be found in the VCD spectra of RNAs as would be expected from the similarity of their structures. The VCD is sequence-dependent. Under the dehydrating conditions studied, poly(dA-dT)poly(dA-dT),poly(dA).poly(dT), and a high-A-T fraction natural DNA had a different bandshape from the other DNAs, which was similar to that of poly(rA).poly(rU). Poly(dG-dC).poly-(dG-dC) did not form an A-form in high-alcohol conditions but instead had a VCD spectrum much like that of its high-salt-induced Z-form. Qualitative differences seen experimentally between A- and B-form DNA VCD were suggested by the differences in the coupled oscillator VCD calculated for the two forms.     

23Na NMR relaxation study of the effects of conformation and base composition on the interactions of counterions with double-helical DNA

NMR relaxation rates (T1(-1) and T2(-1)) have been determined for 23Na in aqueous salt solutions containing various types of helical double-stranded deoxyribonucleic acids. These measurements were performed on three synthetic polynucleotides having different overall conformations, poly-(dA-dT).poly(dA-dT) (alternating B-DNA), poly(dG-dC).poly(dG-dC) at low salt (B-DNA), and Br-poly(dG-dC).Br-poly(dG-dC) (left-handed Z-DNA), and on four types of natural DNA differing in base composition, Clostridium perfringens (26% GC), calf thymus (40% GC), Escherichia coli (50% GC), and Micrococcus lysodeikticus (72% GC). For all types of DNA investigated, except poly(dA-dT).poly(dA-dT), the 23Na NMR spectra measured at 21 degrees C and an applied field of 4.7 T are non-Lorentzian. These non-Lorentzian spectra were analyzed on the basis of the two-state model and the standard theory of nonexponential quadrupolar relaxation processes in order to obtain estimates of the correlation times (tau c) characteristic of the sodium nuclei associated with the various nucleic acids. All of the correlation times estimated in this way are in the range of nanoseconds. The magnitudes of these correlation times show a significant dependence on the overall conformation of the nucleic acid (B vs. Z) but not on its base composition. To investigate the concentration dependence of tau c, sodium or magnesium salts were added to solutions of Br-poly(dG-dC).Br-poly(dG-dC) (Z-DNA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of topotecan,DNA topoisomerase I inhibitor,with double-stranded polydeoxyribonucleotides. 4. Topotecan binds preferably to the GC base pairs of DNA

Interaction of topotecan (TPT) with synthetic double-stranded polydeoxyribonucleotides has been studied in solutions of low ionic strength at pH = 6.8 by linear flow dichroism (LD), circular dichroism (CD), UV-Vis absorption and Raman spectroscopy. The complexes of TPT with poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), poly(dA-dC).poly(dG-dT), poly(dA).poly(dT) and previously studied by us complexes of TPT with calf thymus DNA and coliphage T4 DNA have been shown to have negative LD in the long-wavelength absorption band of TPT, whereas the complex of TPT with poly(dA-dT).poly(dA-dT) has positive LD in this absorption band of TPT. Thus, there are two different types of TPT complexes with the polymers. TPT has been established to bind preferably to GC base pairs because its affinity to the polymers of different GC composition decreases in the following order: poly(dG-dC).poly(dG-dC) > poly(dG).poly(dC) > poly(dA-dC).poly(dG-dT) > poly(dA).poly(dT). The presence of DNA has been shown to shift monomer-dimer equilibrium in TPT solutions toward dimer formation. Several duplexes of the synthetic polynucleotides bound together by the bridges of TPT dimers may participate in the formation of the studied type of TPT-polynucleotide complexes. Molecular models of TPT complex with linear and ring supercoiled DNAs and with deoxyguanosine have been considered. TPT (and presumably all camptothecin family) proved to be a representative of a new class of DNA-specific ligands whose biological action is associated with formation of dimeric bridges between two DNA duplexes.     

DNA-binding affinities and sequence selectivity of quaternary benzophenanthridine alkaloids sanguinarine, chelerythrine, and nitidine

A comparative study on the intercalating binding of sanguinarine, chelerythrine, and nitidine with CT DNA, poly(dG-dC).poly(dG-dC), poly(dA-dT).poly(dA-dT), and seven sequence-designed double-stranded oligodeoxynucleotides has been performed using fluorometric and spectrophotometric techniques, aiming at providing insights into their sequence selectivity for DNA-binding. The results show that both sanguinarine and nitidine bind preferentially to DNA containing alternating GC base pairs [d(TGCGCA)(2)], while chelerythrine exhibits quite distinct sequence selectivity from sanguinarine, which shows a high specificity for DNA containing contiguous GC base pairs [5'-TGGGGA-3'/3'-ACCCCT-5'].     

Kinetics of the proton-deuteron exchange at position H8 of adenine and guanine in DNA.

Proton-NMR has been used to determine the activation energies and pre-exponential factors for the deuterium exchange of AH8 in poly(dA-dT).poly(dA-dT), and for GH8 in poly(dG-dC).poly(dG-dC). No simple relationship between the kinetic parameters and molecular conformation was found. By addition of 4.5 M NaCl a transition from the B to the Z conformation was induced for poly(dG-dC).poly(dG-dC), and an increased exchange rate was observed. The exchange rate for poly(dA-dT).poly(dA-dT) also increased below 64 degrees C, and a significant decrease in activation energy on addition of 4.5 M NaCl was observed. The exchange rates at T = 55.8 degrees C were also measured for the AH8 and GH8 in random sequence calf thymus DNA. From the difference in exchange rates, a method of preferential labeling of either the AH8 or the GH8 in high molecular weight DNA is evaluated.     

Uracil in deoxyribonucleotide polymers reduces their template-primer activity for E. coli DNA polymerase I.

The physical and biochemical properties of two pairs of synthetic DNA template-primers were investigated. The copolymer poly(dA-dU) . poly(dA-dU) and the homopolymer duplex poly(dA). poly(dU) were characterized by a lower Tm and by a higher buoyant density value than the respective thymine polynucleotides poly(dA-dT) . poly(dA-dT) and poly(dA) . poly(dT). The polymerizing and the primer terminus adding reactions of a homogenous E. coli DNA polymerase I preparation, as measured by incorporation of [3H]dAMP into the acid-insoluble fraction, were significantly poorer with uracil-containing template-primers than with thymine templates. Moreover, the uracil-containing polynucleotides inhibited the polymerizing activity of DNA polymerase I to a greater extent than the thymine polynucleotides, when the enzymatic activity was investigated with a dATP/dTTP/dUTP-free incorporation system making use of poly(dI-dC) . poly(dI-dC) as the template-primer.     

Sequence-dependent mechanics of single DNA molecules

Atomic force microscope-based single-molecule force spectroscopy was employed to measure sequence-dependent mechanical properties of DNA by stretching individual DNA double strands attached between a gold surface and an AFM tip. We discovered that in lambda-phage DNA the previously reported B-S transition, where 'S' represents an overstretched conformation, at 65 pN is followed by a nonequilibrium melting transition at 150 pN. During this transition the DNA is split into single strands that fully recombine upon relaxation. The sequence dependence was investigated in comparative studies with poly(dG-dC) and poly(dA-dT) DNA. Both the B-S and the melting transition occur at significantly lower forces in poly(dA-dT) compared to poly(dG-dC). We made use of the melting transition to prepare single poly(dG-dC) and poly(dA-dT) DNA strands that upon relaxation reannealed into hairpins as a result of their self-complementary sequence. The unzipping of these hairpins directly revealed the base pair-unbinding forces for G-C to be 20 +/- 3 pN and for A-T to be 9 +/- 3 pN.     

Enzymatic properties of de novo-type mouse DNA (cytosine-5) methyltransferases

We have purified GST-fused recombinant mouse Dnmt3a and three isoforms of mouse Dnmt3b to near homogeneity. Dnmt3b3, an isoform of Dnmt3b, did not have DNA methylation activity. Dnmt3a, Dnmt3b1 or Dnmt3b2 showed similar activity toward poly(dG-dC)-poly(dG-dC) for measuring de novo methylation activity, and toward poly(dI-dC)-poly(dI-dC) for measuring total activity. This indicates that the enzymes are de novo-type DNA methyltransferases. The enzyme activity was inhibited by NaCl or KCl at concentrations >100 mM. The kinetic parameter, K_m^AdoMet, for Dnmt3a, Dnmt3b1 and Dnmt3b2 was 0.4, 1.2 and 0.9 µM when poly(dI-dC)-poly(dI-dC) was used, and 0.3, 1.2 and 0.8 µM when poly(dG-dC)-poly(dG-dC) was used, respectively. The K_m^DNA values for Dnmt3a, Dnmt3b1 and Dnmt3b2 were 2.7, 1.3 and 1.5 µM when poly(dI-dC)-poly(dI-dC) was used, and 3.5, 1.0 and 0.9 µM when poly(dG-dC)-poly(dG-dC) was used, respectively. For the methylation specificity, Dnmt3a significantly methylated CpG >> CpA. On the other hand, Dnmt3b1 methylated CpG > CpT ≥ CpA. Immuno-purified Dnmt3a, Myc-tagged and overexpressed in HEK 293T cells, methylated CpG >> CpA > CpT. Neither Dnmt3a nor Dnmt3b1 methylated the first cytosine of CpC.     

Mechanical stability of single DNA molecules

Using a modified atomic force microscope (AFM), individual double-stranded (ds) DNA molecules attached to an AFM tip and a gold surface were overstretched, and the mechanical stability of the DNA double helix was investigated. In lambda-phage DNA the previously reported B-S transition at 65 piconewtons (pN) is followed by a second conformational transition, during which the DNA double helix melts into two single strands. Unlike the B-S transition, the melting transition exhibits a pronounced force-loading-rate dependence and a marked hysteresis, characteristic of a nonequilibrium conformational transition. The kinetics of force-induced melting of the double helix, its reannealing kinetics, as well as the influence of ionic strength, temperature, and DNA sequence on the mechanical stability of the double helix were investigated. As expected, the DNA double helix is considerably destabilized under low salt buffer conditions (相似文献   

Molecular recognition of DNA by small molecules: AT base pair specific intercalative binding of cytotoxic plant alkaloid palmatine

The base dependent binding of the cytotoxic alkaloid palmatine to four synthetic polynucleotides, poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) was examined by competition dialysis, spectrophotometric, spectrofluorimetric, thermal melting, circular dichroic, viscometric and isothermal titration calorimetric (ITC) studies. Binding of the alkaloid to various polynucleotides was dependent upon sequences of base pairs. Binding data obtained from absorbance measurements according to neighbour exclusion model indicated that the intrinsic binding constants decreased in the order poly(dA).poly(dT)>poly(dA-dT).poly(dA-dT)>poly(dG-dC).poly(dG-dC)>poly(dG).poly(dC). This affinity was also revealed by the competition dialysis, increase of steady state fluorescence intensity, increase in fluorescence quantum yield, stabilization against thermal denaturation and perturbations in circular dichroic spectrum. Among the polynucleotides, poly(dA).poly(dT) showed positive cooperativity at binding values lower than r=0.05. Viscosity studies revealed that in the strong binding region, the increase of contour length of DNA depended strongly on the sequence of base pairs being higher for AT polymers and induction of unwinding-rewinding process of covalently closed superhelical DNA. Isothermal titration calorimetric data showed a single entropy driven binding event in the AT homo polymer while that with the hetero polymer involved two binding modes, an entropy driven strong binding followed by an enthalpy driven weak binding. These results unequivocally established that the alkaloid palmatine binds strongly to AT homo and hetero polymers by mechanism of intercalation.     

DNA solution conformation via infrared circular dichroism: experimental and theoretical results for B-family polymers

Infrared (vibrational) circular dichroism (VCD) has been observed for the DNA models d(CG)5, poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), poly(dA-dT).poly(dA-dT), and poly(dA).poly(dT) in the B-conformation in buffered, aqueous solution. The observed results are quantitatively interpreted in terms of the exciton model for coupled carbonyl stretching vibrational states.     

Conformational transitions of alternating purine-pyrimidine DNAs in perchlorate ethanol solutions.

Conformational transitions of poly(dA-dC).poly(dG-dT), poly(dA-dT).poly(dA-dT), and other alternating purine-pyrimidine DNAs were studied in aqueous ethanol solutions containing molar concentrations of sodium perchlorate, which is a novel solvent stabilizing non-B duplexes of DNA. Using CD and UV absorption spectroscopies, we show that this solvent unstacks bases and unwinds the B-forms of the DNAs to transform them into the A-form or Z-form. In the absence of divalent cations poly(dA-dC).poly(dG-dT) can adopt both of these conformations. Its transition into the Z-form is induced at higher salt and lower ethanol concentrations, and at higher temperatures than the transition into the A-form. Submillimolar concentrations of NiCl2 induce a highly cooperative and slow A-Z transition or Z-Z' transition, which is fast and displays low cooperativity. Poly(dA-dT).poly(dA-dT) easily isomerizes into the A-form in perchlorate-ethanol solutions, whereas high perchlorate concentrations denature the polynucleotide, which then cannot adopt the Z-form. At low temperatures, however, NiCl2 also cooperatively induces the Z'-form in poly(dA-dT).poly(dA-dT). Poly(dI-dC).poly(dI-dC) is known to adopt an unusual B-form in low-salt aqueous solution, which is transformed into a standard B-form by the combination of perchlorate and ethanol. NiCl2 then transforms poly(dI-dC).poly(dI-dC) into the Z'-form, which is also adopted by poly(dI-br5dC).poly(dI-br5dC).