PubMed Health⌕ Search

Biomedical subjects

J Sági

Publications and source records attributed to J Sági.

At least 37 records · Page 2Linked to original sources
PubMed1993

Conformational isomerizations of poly(dA-dT) are dramatically influenced by a substitution of a minor amount of adenine by purine or amino2purine.

We have synthesized poly(dA,dPu-dT) and poly(dA,n2dPu-dT) containing, respectively, 5.7% of purine and 7.4% of amino2purine in place of adenine to demonstrate that these apparently negligible perturbations of the primary structure have dramatic consequences for the polynucleotide conformational isomerizations. The replacement of adenine by amino2purine, preserving the number of hydrogen bonds between the complementary bases, has a stronger effect on the polynucleotide conformational isomerizations than the replacement with purine that is bound only by a single hydrogen bond to thymine. Nevertheless, poly(dA,dPu-dT) forms a more thermostable duplex than poly(dA,n2dPu-dT). Furthermore the few amino2purines in poly(dA,n2dPu-dT) inhibit its isomerization into X-DNA, stabilize but modify A-DNA and stabilize Z-DNA. Kinetics of the B-Z transition of poly(dA,n2dPu-dT) is fast to indicate that the amino groups in the double helix minor groove substantially decrease the kinetic barrier between B- and Z-DNA. On the other hand, the replacement of adenine by purine destabilizes both Z-DNA and A-DNA, and the destabilization of X-DNA is weaker than with amino2purine. A-form and B-form perhaps coexist in poly(dA,dPu-dT) at high concentrations of ethanol.

Adenine↗
PubMed1993

[Incorporation of the antiherpetic 5-isopropyl-2'-deoxyuridine into a synthetic DNA and the consequence of incorporation on structure and functions of the DNA].

5-Isopropyl-2'-deoxyuridine is the active ingredient of Hevizos ointment, a commercially available drug in Hungary, applied on the local treatment of patient with herpes labialis, zoster and progenitalis. The 5'-triphosphate derivative of the compound was incorporated by a DNA polymerase enzyme into a synthetic DNA of poly(dA-dT) type in order to study possible alterations in the structure and bioorganic functions of the DNA, in comparison with unmodified poly(dA-dT). Alterations observed by spectroscopic, electron microscopic and biochemical methods refer to the formation of stable hairpins protruding from the DNA duplex, which may be responsible for the impairment of the integrity of the DNA structure. This, in turn, may negatively influence normal biochemical functions of a DNA.

Antiviral Agents↗
PubMed1992

Structures of poly(dA-dT, ip5dU) containing various small amounts of the antiherpetic 5-isopropyl-2'-deoxyuridine.

Three different concentrations of the antiherpetic agent 5-isopropyl-2'-deoxyuridine (ip5dU) were introduced into the synthetic DNA poly(dA-dT) to analyze resulting copolymers by electron microscopy, UV absorption and CD spectroscopy. The poly(dA-dT, ip5dU) containing 1.3 and 4.3% ip5dU did not much differ from the parent poly(dA-dT) but poly (dA-dT, ip5dU) with 7.1% ip5dU behaved in an unusual way. Results are explained by the notion that if bulky isopropyls occur sufficiently close to each other then stable hairpins protruding from the double helix are formed, presumably to accommodate the ip5dU-s into the loops.

Antiviral Agents↗
PubMed1992

FT-IR spectroscopic study of the poly(amino2dA-dT) duplex in Mg(2+)-containing solution and in films.

The alternative structures of the synthetic poly(amino2dA-dT) duplex have been studied using infrared spectroscopy in films and in solution (D2O and H2O) in the presence and in the absence of magnesium salt. In solution without magnesium salt, the polynucleotide exists in a B genus conformation with some of the sugar puckers possibly in the C3'-endo/anti geometry. In magnesium-containing solution (66 mM MgCl2), however, we report infrared spectra of Mg(2+)-poly(amino2dA-dT) which have characteristic marker bands of the A form. Film samples in 70% relative humidity (RH) give similar infrared spectra to those of the polynucleotide obtained using Mg2+. Thus, when analyzed in comparison with previously reported infrared spectra of other oligo and polynucleotides, our data show that double helical poly(amino2dA-dT) goes into the same (or very closely related) conformation in dehydrated films as in solutions containing Mg2+.

Deuterium↗
PubMed1991

Transitions of poly(dI-dC), poly(dI-methyl5dC) and poly(dI-bromo5dC) among and within the B-, Z-, A- and X-DNA families of conformations.

It is shown, using circular dichroism spectroscopy, that poly(dI-dC) is capable to isomerize into both Z-DNA and A-DNA in concentrated NaCl + NiCl2 and trifluoroethanol solutions, respectively. This polynucleotide also undergoes a cooperative, two-state transition in ethanol into a structure which most probably is a canonical B-DNA. This implies that the conformation of poly(dI-dC) is unusual in low-salt aqueous solution. The canonical B-DNA is also adopted by poly(dI-methyl5dC) in trifluoroethanol while this polynucleotide adopts Z-DNA not only in NaCl + NiCl2 but also in the presence of MgCl2. Poly(dI-methyl5dC) partially adopts X-DNA in concentrated CsF and mainly ethanolic solutions. Poly(dI-bromo5dC) isomerizes into Z-DNA not only in concentrated NaCl even in the absence of NiCl2 but also in concentrated MgCl2. This polynucleotide transforms between two distinct variants of Z-DNA in ethanol or trifluoroethanol solutions.

Circular Dichroism↗
PubMed1991

Destabilization of the duplex and the high-salt Z-form of poly(dG-methyl5dC) by substitution of ethyl for the 5-methyl group.

The B-to-Z conformational transition of poly(dG-dC) is highly promoted by 5-methyl substitution of the dC moiety, i.e. in poly(dG-methyl5dC). By the synthesis of a new poly(dG-dC) analogue, poly(dG-ethyl5dC), the effect of a longer alkyl-chain substituent of dC on structure and conformation has been studied with ultraviolet absorption melting profiles and circular dichroism spectroscopy. The 5-ethyl substituent in poly(dG-ethyl5dC) destabilizes the duplex structure against thermal denaturation compared with both poly(dG-methyl5dC) and poly(dG-dC). C.d. studies also reveal that for the high-salt B-Z transition of poly(dG-ethyl5dC) a higher NaCl concentration is required than for that of poly(dG-methyl5dC), although much lower than for poly(dG-dC). However low-salt Z-DNA in poly(dG-ethyl5dC) shows unique features, e.g. it needs no divalent cations to be stable. The low-salt B-Z transition of poly(dG-ethyl5dC) can also be observed by the absorption-temperature melting profile, in contrast to both poly(dG-methyl5dC) and poly(dG-dC). The effects of MgCl2 concentration, temperature, acid pH and trifluorethanol on the conformation of poly(dG-ethyl5dC) have also been determined.

Circular Dichroism↗
PubMed1991

Alkyl substituent in place of the thymine methyl group controls the A-X conformational bimorphism in poly(dA-dT).

Circular dichroism studies of a family of poly(dA-y5dU) polynucleotides (y = H, methyl, ethyl, propyl, butyl or pentyl) were conducted in water-alcohol solutions containing sodium or cesium counterions. The polynucleotides denatured or adopted A- or X-DNA double helices depending on the concentration and type of alcohol, type of counterions and the length of the aliphatic substituent in place of the thymine methyl group. Short aliphatic substituents and sodium cations favored A-DNA while long aliphatic substituents and cesium cations promoted X-DNA. This study demonstrates delicacy of the conformational equilibrium of poly(dA-dT) between the A- and X-DNA double helices which depends on both intramolecular and intermolecular factors.

Alkylation↗
PubMed1990

Biochemical properties of oligo [(+)-carbocyclic-thymidylates] and their complexes.

We report here spectroscopic and biochemical data of a novel series of sugar-modified oligodeoxy-nucleotides, the carbocyclic oligothymidylates, c(dT)3-20. In c(dT)n a methylene group has been substituted for the oxygen atom of the deoxyribose ring of the natural thymidylate unit. c(dT)10-20 form helical structures, in contrast with oligothymidylates or poly(dT), based on absorbance versus temperature melting profiles. Secondary structure of c(dT)n, where n greater than 10 is assumed to be double helix. In addition to this, c(dT)n forms as a stable duplex with complementary poly(dA) as does parent (dT)n. On the other hand, c(dT)n-containing oligo/poly duplex is nearly inactive either as a template or as a primer in various DNA polymerase systems, and c(dT)n inhibits DNA replication as well. c(dT)n can efficiently be extended by terminal transferase and shows an increased nuclease stability compared to (dT)n. Base-pairing ability and nuclease stability of c(dT)n suggest that (+)-carbocyclic nucleoside-containing oligomers could be new potential antisense oligodeoxynucleotides.

Absorption↗
PubMed1990

Conformational isomerizations of the poly(dA-dT) and poly(amino2dA-dT) duplexes involving the unusual X-DNA double helix: a fourth derivative spectrophotometric study.

Fourth derivative spectrophotometry has been applied to monitor conformational isomerizations of polynucleotides for the first time. The transitions studied have been the B-A and A-X isomerizations of poly(dA-dT) and the B-X one of poly(amino2dA-dT). Parameters obtained from the fourth derivative spectra have been used to follow these conformational changes. The A form of poly(dA-dT) has been characterized by a new fourth derivative peak at 293.0 nm which can be associated to interstrand adenine-adenine interactions. Furthermore, some of the fourth derivative peaks in the long wavelength region (270-310 nm) can be related to stacking interactions present in the polynucleotide double helices. The tentative assignment of these peaks, particularly that at 299.0 nm in the derivative spectra of poly(amino2dA-dT), to n----pi electronic transitions is discussed.

Isomerism↗
PubMed1990

Replication, transcription and nuclease digestion of the unusual X-DNA double helix of poly(amino2dA-dT).

The alternating copolymer poly(amino2dA-dT) isomerizes into the unusual X-DNA double helix at low-salt aqueous conditions (Vorlicková et al., J. Biomolec. Struct. Dyn. 6, 503-510 (1988)). This observation allowed us to start studies on how the X-DNA is recognized, copied and hydrolyzed by various enzymes. In the present paper X-DNA replication, transcription and digestion by various polymerases and nucleases, respectively, are examined and compared to appropriate controls. It is found that X-DNA is a poor primer-template for DNA synthesis by the E. coli Klenow DNA polymerase (12% of the activity observed with B-DNA), the Micrococcus luteus DNA polymerase I (25%) and the AMV reverse transcriptase (51%). In contrast, X-DNA is a better template by 74% than B-DNA for calf thymus DNA polymerase alpha. For transcription by E. coli RNA polymerase enzyme poly(amino2dA-dT) did not serve as a template at all in either B or X conformation. Poly(amino2dA-dT) in its B form proved to be much more stable than poly(dA-dT) against hydrolysis by pancreatic DNase and snake venom phosphodiesterase. Formation of the X conformation in poly(amino2dA-dT) decreased this large difference in nuclease stability.

DNA Replication↗
PubMed1989

Recognition and use of the unusual X-DNA as a primer-template by Klenow DNA polymerase enzyme.

Based on CD spectra, 2-amino-2'-deoxyadenosine-containing synthetic alternating DNA, poly(amino2dA-dt) undergoes a conformational transition from a B-form to a non-Z zig-zag form of DNA, called X, even under conditions where enzymes can work. Kinetic parameters of the E. coli Klenow DNA polymerase enzyme-catalyzed copying of both the B- and X-forms of poly(amino2dA-dT) have been determined. Binding affinity of X-DNA to the enzyme proved to be even higher than that of the B-DNA; primer-chain extension of X-poly(amino2dA-dT) was however hindered as compared to its B-form. This differential utilization of X-DNA versus B-DNA by a DNA polymerase is an in vitro enzymatic evidence of an unusual DNA conformation.

Circular Dichroism↗
PubMed1989

Divalent cations are not required for the stability of the low-salt Z-DNA conformation in poly(dG-ethyl5dC).

It is demonstrated that poly(dG-ethyl5dC) adopts Z form in low-salt solution like poly(dG-methyl5dC). Its existence is, however, not contingent on the presence of divalent cations in the polynucleotide solution. The Z form is transformed into B form below room temperature. The arising B form cannot be transformed back into Z form by millimolar MgCl2 concentrations. On the contrary, the addition of MgCl2 at room temperature converts the low-salt Z form of poly(dG-ethyl5dC) into B form. It follows from the results that Z form is a stable DNA conformation not only at high but even at low ionic strengths.

Cations, Divalent↗
PubMed1988

Conformation of the synthetic DNA poly(amino2dA-dT) duplex in high-salt and aqueous alcohol solutions.

It has previously been demonstrated by other workers that the duplex of a synthetic DNA poly(amino2dA-dT) undergoes a salt-induced conformational isomerization. We show in the present work using circular dichroism that the same isomerization is induced in poly(amino2dA-dT) by various alcohols. The isomerization was originally identified as the B-to-Z and then B-to-A conformational transition of DNA but we demonstrate that the high-salt or alcohol conformation of poly (amino2dA-dT) is the non Z-DNA zig-zag double helix we have previously observed with poly(dA-dT) and called X-DNA. X-DNA is a cesium cation specific conformation of poly(dA-dT) while no similar cation specificity is observed with poly(amino2dA-dT). Thus it appears that the extra amino group attached to A and cesium cations make the same thing; they probably dehydrate the double helix minor groove and relieve its conformational variability. Poly(amino2dA-dT) is exceptionally stable in X-DNA and conditions inducing it are mild, which opens the door to assess its molecular structure.

Circular Dichroism↗
PubMed1987

Incorporation of the carbocyclic analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate into a synthetic DNA.

The carbocyclic analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine, C-BVDU, is a very potent and selective anti-herpes-virus compound. In order to synthesize and study the properties of a DNA that contains C-BVDU, the 5'-triphosphate, C-BVDUTP was prepared and evaluated as a potential substrate of the E. coli Klenow DNA polymerase enzyme. Although C-BVDUTP proved to be a very poor substrate also of this enzyme, it could be incorporated up to 3.6% into the synthetic DNA, poly(dA-dT, C-BVDU). This level of substitution decreased significantly the template activity for DNA and RNA polymerases, as compared to that of poly(dA-dT).

Bromodeoxyuridine↗
PubMed1987

Substrate specificity of DNA polymerases. I. Enzyme-catalysed incorporation of 5-'1-alkenyl)-2'-deoxyuridines into DNA.

A series of (E)-5-(1-alkenyl)-dUTPs as well as 5-vinyl-and (Z)-5-(1-propenyl)-dUTP have been synthesized to study steric requirements in DNA polymerase reactions. Experiments were carried out in E. coli DNA polymerase I Klenow fragment enzyme system. Substrates were characterized by KM and Vmax-values, initial incorporation rates as well as by total extent of incorporation of the analogues into poly(dA-dT) as a template-primer. Incorporation of the analogues could be best correlated with Vmax-values as well as the very similar initial incorporation rate values. Reactivity (Vmax/KM) showed no correlation with the extent of incorporation. 5-Vinyl-dUTP proved to be as good a substrate of the enzyme as dTTP, whereas (E)-5-(1-heptenyl)-and (E)-5-(1-octenyl)-dUTPs were very poor substrates, their incorporation was strongly limited and they also proved to be very efficient inhibitors of DNA replication, as shown by Ki-values. Substrate specificity of the Klenow enzyme can be explained by the steric hindrance of C-5 substituent, by the "orientational steric substituent effect" concept.

Alkenes↗
PubMed1987

Carbocyclic analogues of dTTP and UTP: properties in polymerase enzyme-catalyzed reactions.

Racemic carbocyclic analogues of dTTP [(+/-)-C-dTTP] and its ribo counterpart, 5-methyl-UTP [(+/-)-C-m5UTP] were synthesized and examined, in comparison with dTTP and UTP (and m5UTP), as potential substrates of E. coli DNA and RNA polymerases, respectively. Unexpectedly, only a very low (terminal) incorporation of C-dTMP into DNAs of different structure was observed, C-dTTP did not serve as a substrate for chain elongation by the Klenow DNA polymerase. Inhibition of DNA replication was, however, observed in the presence of (+/-)-C-dTTP. The UTP analogue, (+/-)-C-m5UTP proved neither a substrate nor an inhibitor of the RNA polymerase enzyme.

DNA-Directed DNA Polymerase↗