Biomedical subjects

J Sági

Publications and source records attributed to J Sági.

At least 19 recordsLinked to original sources

PubMed2001

15-mer DNA duplexes containing an abasic site are thermodynamically more stable with adjacent purines than with pyrimidines.

Abasic site (AP)-containing duplexes, with flanking adenine (A) or cytosine (C) bases, were shown to be more stable with flanking A than with C bases [Sági, J., Hang, B., and Singer, B. (1999) Chem. Res. Toxicol. 12, 917-923]. We investigated whether the lower-magnitude destabilization by an AP site, with A neighbors, is a general effect of the purine versus the pyrimidine neighbors. Duplex stability, as compared to that of the corresponding control duplexes, was markedly decreased by the incorporation of the AP site (x) opposite any of the four bases. However, for the duplexes containing T, A, or C opposite the AP site, replacement of the symmetric doublet flanking pyrimidine bases with purines resulted in a smaller destabilization effect. The average stabilizing effect of the symmetric doublet purine neighbors of an AP site opposite T, A, or C bases was 3.2 degrees C (DeltaT(m)) and 1.3 kcal/mol (DeltaDeltaG degrees (37)) compared to those of pyrimidine neighbors. In contrast, a G.AP pair reduced or eliminated the differential effect of the neighbors. Using unrestrained molecular dynamics, it was shown that for the duplexes containing T opposite the AP site, with doublet pyrimidine neighbors, there was a larger magnitude of curvature around the lesion site than for the duplexes with the purines flanking the AP site. Purines flanking the AP site tend to shift toward each other, creating overlap, in contrast to the flanking pyrimidines. This indicates the possibility of stacking between purine bases at the AP site and can be the reason for the observed smaller thermodynamic destabilization of the duplexes with the AAxAA and GGxGG central sequences, as compared to those with TTxTT and CCxCC sequences. This work showed that for an AP site the GC content is not the only determinant of duplex stability, but rather is influenced more by whether purines or pyrimidines flank the AP site.

Adenine↗

PubMed2000

Differential destabilization of the DNA oligonucleotide double helix by a T.G mismatch, 3,N(4)-ethenocytosine, 3,N(4)-ethanocytosine, or an 8-(hydroxymethyl)-3,N(4)-ethenocytosine adduct incorporated into the same sequence contexts.

The T.G mismatch and the exocyclic adduct 3,N(4)-ethenocytosine (epsilonC) are repaired by the same enzyme, the human G/T(U) mismatch-DNA glycosylase (TDG). This enzyme removes the T, U, or epsilonC base from duplex DNA. The rate of cleavage was found to differ with the lesion and was also affected by neighbor sequences [Hang, B., Medina, M., Fraenkel-Conrat, H., and Singer, B. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 13561-13566]. Since sequence influences duplex stability, we determined the thermodynamic stability of T.G and epsilonC-containing 15-mer duplexes in which the bases flanking the lesion were systematically varied. The duplexes contained central 5'-TTXTT, 5'-AAXAA, 5'-CCXCC, or 5'-GGXGG sequences, where X is T, epsilonC, or two closely related structural derivatives of epsilonC: 3,N(4)-ethanocytosine (EC) and 8-(hydroxymethyl)-epsilonC (8-HM-epsilonC). Each of the four lesions, incorporated opposite G, decreased both the thermal (T(m)) and thermodynamic stability (DeltaG degrees (37)) of the 15-mer control duplexes. On the basis of the T(m) and DeltaG degrees (37) values, the order of destabilization of the TTXTT sequence in 15-mer duplexes was as follows: 8-HM-epsilonC > EC > epsilonC > T.G. The DeltaT(m) values range from -15.8 to -9.5 degrees C when C(t) = 8 microM. Duplexes with flanking AA or TT neighbors were more destabilized, by an average of 2 degrees C, than those with flanking GG or CC neighbors. The base opposite the modified base also influenced duplex stability. Within the TT context, of the four changed bases opposite the adducts, C had the greatest destabilizing effect, up to -18.4 degrees C. In contrast, a G opposite an adduct was generally the least destabilizing, and the smallest value was -3. 0 degrees C. Destabilizations were enthalpic in origin. Thus, this work shows that independently changing the modified base, the sequence, or the base opposite the lesion each affects the stability of the duplex, to significantly varying extents. The potential contribution of the thermodynamic stability to repair efficiency is discussed.

Base Pair Mismatch↗

PubMed2000

A structure-function study of nucleic acid-fluorenone complexes.

Several 2.7-bis-[(dialkylamino)-acetylamino]-fluoren-9-one derivatives (fluoramides) were synthesized as analogues of the DNA binding compound tilorone (2,7-bis[(diethylamino)-ethoxy]-fluoren-9-one). Previous studies showed the drugs to induce cytokines and inhibit reverse transcription. Here, their binding to DNA was evaluated using UV and circular dichroism studies. Like tilorone, the fluoramides derivatives also intercalate resulting in increased Tm values and new CD signatures. A preference to alternating A-T and G-C sequences was detected; only minor interaction to homologous sequences was observed. Moreover, no upper limit in the drug/DNA ratio was found, testing limit being the precipitation of the drug. However, surface plasmon resonance (SPR) studies of tilorone and 2,7-bis-[(dipropylamino)-acetyl-amino]-fluoren-9-one, indicate an astonishing drug/base pair ratio (r > 1), which point to a multitude of interactions under SPR conditions. Molecular modeling calculations, where the geometries of the complexes optimized under the assumption of intercalative and multitude of suprahelical arrangements, rationalize the observations. Based on the thermodynamic and biological studies, a structure-function model is proposed.

Animals↗

PubMed2000

Sequence-dependent conformational perturbation in DNA duplexes containing an epsilonA.T mismatch using molecular dynamics simulation.

Previous experiments from this laboratory showed that 1, N:(6)-ethenoadenine (epsilonA) in 15mer DNA oligonucleotide duplexes with GGepsilonAGG and CCepsilonACC central sequences is repaired 3-5-fold more efficiently than in duplexes containing AAepsilonAAA and TTepsilonATT central sequences. This sequence dependence in repair rates appeared to correlate with the observed thermodynamic stability of these duplexes [Hang et al. (1998) J. Biol. Chem., 273, 33406-33413]. In the present work, unrestrained molecular dynamics was used to evaluate the sequence-dependent structural features of these duplexes. Explicit solvent and the particle mesh Ewald method were applied for the accurate representation of the electrostatic interactions. The differences observed in the axis- and intra-base pair parameters were primarily localized at the epsilonA*T mismatch in all sequences and indicate conformational diversity between the structures. However, all four structures remained in the B-conformational family. In the tip, tilt and propeller twist parameters for the five central base pairs, larger perturbations were found for the two duplexes with epsilonA flanked by A or T bases than for duplexes with epsilonA flanked by G or C bases. As a result of these perturbations, the average global curvature of the AAepsilonAAA and TTvarepsilonATT DNA duplexes was larger by approximately 12 degrees than that of the duplexes with the GGepsilonAGG and CCepsilonACC central sequences. The observed conformational differences between the duplexes containing A or T and G or C neighbors of epsilonA may contribute to the observed differential enzymatic repair of the same sequences.

Adenine↗

PubMed1999

Sequence-dependent repair of synthetic AP sites in 15-mer and 35-mer oligonucleotides: role of thermodynamic stability imposed by neighbor bases.

We previously reported that 15-mer oligonucleotides with a central 1, N(6)-epsilonA were cleaved by alkylpurine-DNA N-glycosylase as a function of T(m), modulated by neighbor bases [Hang, B., Sági, J., and Singer, B. (1998) J. Biol. Chem. 273, 33406-33413]. This type of investigation has now been extended to cleavage by Escherichia coli endonuclease IV of a centrally placed synthetic AP site using both 15-mer and 35-mer duplexes. In 15-mers, the triplet sequences adjunct to the central AP site greatly affected the thermodynamic stability. The repair rate paralleled the thermal stability since endonuclease IV requires a double-stranded substrate. When the AP site-containing duplexes were 35-mers, there was also a general correlation between the thermostability and cleavage efficiency. However, the difference in the cleavage rates between different sequences was much less than with the 15-mers. Since the 35-mers were more than 96% annealed, this difference presumably results from local stability and structure adjacent to the AP site. These results suggest that under enzyme limiting conditions or overproduction of AP sites, sequence-dependent differential repair could occur in vivo.

Amino Acid Sequence↗

PubMed1999

Thermal destabilization of DNA oligonucleotide duplexes by exocyclic adducts on adenine or cytosine depends on both the base and the size of adduct.

Numerous carcinogens or their bifunctional metabolites modify DNA bases by forming additional exocyclic rings on the base moiety. These modifications form exocyclic rings between N1 and N6 of dA, N3 and N4 of dC or N1 and N2 of dG, as well as the N2 and N3 of dG. This study focuses on the reaction products of dA and dC with chloroacetaldehyde, bis-chloroethyl nitrosourea and para-benzoquinone, which form etheno, ethano and para-benzoquinone derivatives, respectively. The three dC adducts and three dA adducts were each incorporated site-specifically into 25-nucleotide-long deoxyoligonucleotides. All duplexes with a single modified dA or dC adduct opposite the normal complement showed decreased thermal stability, as compared with the unmodified control duplex. The destabilizations ranges from -2 degrees C to -13 degrees C, depending on saturation, the size of the adduct and the nature of the base. Energy-minimized molecular models of the duplexes illustrate various degrees of distortions by the adducts, the para-benzoquinone adducts showing the greatest distortion.

Base Sequence↗

PubMed1998

Correlation between sequence-dependent glycosylase repair and the thermal stability of oligonucleotide duplexes containing 1, N6-ethenoadenine.

Previous experiments on DNA sequence context reported that base modification, replication, and repair are affected by the nature of neighbor bases. We now report that repair by mammalian alkylpurine-DNA-N-glycosylases (APNG) of 15-mer oligonucleotides with a central 1,N6-ethenoadenine (epsilonA), flanked by 5' and 3' tandem bases, is also highly sequence dependent. Oligonucleotides with the central sequences -GGepsilonAGG- or -CCepsilonACC- are repaired 3-5-fold more efficiently than those containing -AAepsilonAAA- or -TTepsilonATT- when using human or mouse APNG. Melting curves of the same duplexes showed that oligomers with G.C/C. G neighbors were less denatured than those with A.T/T.A neighbors at 37 degreesC. This sequence-dependent difference in denaturation correlates with the relative thermodynamic stability of oligomers with G.C/C.G or A.T/T.A neighbors. The dependence of repair on thermal stability was confirmed by enzyme reactions performed over 0-45 degreesC. Under these conditions, repair of epsilonA flanked by G.C/C.G was dramatically increased at 37 degreesC with continuous increase up to 45 degreesC, in contrast to that with flanking A.T/T. A pairs, which was in agreement with the degree of denaturation of these duplexes. These results indicate that the thermodynamic stability conferred by base pairs flanking epsilonA plays an essential role in maintaining the integrity of the duplex structure which is necessary for repair.

Adenine↗

PubMed1998

A single cyclic p-benzoquinone adduct can destabilize a DNA oligonucleotide duplex.

p-Benzoquinone (p-BQ), a stable metabolite of the human carcinogen benzene, forms two-ring benzetheno exocyclic base adducts with C, A, and G bases in DNA. As a part of a project for studying the biological effect of the p-BQ adducts, we report here on the first biophysical characterization of oligodeoxyribonucleotide duplexes containing a single site-specific p-BQ-C, using thermal denaturation and circular dichroism (CD). We find that the thermal and thermodynamic stabilities of the control duplex are reduced by p-BQ-C. The Tm value decreases by 12.6 degrees C at the duplex concentration of 1.5 microM and the Delta G o by 10.2 kcal/mol. The latter was determined from the concentration dependence of the Tm values. The destabilization has little dependence on the nature of the opposite base. This reduction is higher than that of the single base mismatches studied (-4.9 to -7.9 kcal/mol) and is close to that observed with an adjacent double mismatch-containing duplex (-11.3 kcal/mol). The overall B-conformation of the duplex with a p-BQ-C is, however, only slightly altered, relative to the parent duplex, as shown by CD spectra. The p-BQ-C-containing duplex has been found recently to be a good substrate for the major human AP endonuclease as compared to an abasic site-containing duplex [Hang, B., et al. (1997) Biochemistry 36, 15411-15418]. We now find that the thermodynamic properties and the localized conformational changes of a p-BQ-C-containing duplex are apparently related to those reported for a duplex containing an abasic site.

Benzoquinones↗

PubMed1998

Differential cleavage of oligonucleotides containing the benzene-derived adduct, 1,N6-benzetheno-dA, by the major human AP endonuclease HAP1 and Escherichia coli exonuclease III and endonuclease IV.

We report here that the newly synthesized DNA adduct, 1,N6-benzetheno-dA (pBQ-dA), in defined oligonucleotides [Chenna and Singer, Chem. Res. Toxicol., 8, 865-874], is a substrate for the major human AP endonuclease, HAP1, and the Escherichia coli AP endonucleases, exonuclease III and endonuclease IV. The mechanism of cleavage is identical to that reported previously for 3,N4-benzetheno-dC (pBQ-dC) and leads to a phosphodiester bond cleavage 5' to the adduct. There are, however, significant differences in the rate of cleavage of this adduct by these enzymes. The two bacterial AP endonucleases are both much more efficient than the human repair enzyme. In addition, using two random oligodeoxynucleotide sequences containing a single pBQ-dA, exonuclease III and endonuclease IV are similarly active, while HAP1 shows a distinct sequence preference of approximately 10-fold in efficiency of cleavage. The repair of this adduct by the three recombinant enzymes is further confirmed by using both active site mutant HAP1 proteins and by E.coli mutant strains lacking exonuclease III and/ or endonuclease IV. This sequence-dependent repair of pBQ-dA by HAP1 may play an important role in modulating benzene-induced carcinogenesis.

Benzimidazoles↗

PubMed1997

Inhibition of IgE production by epsilon (epsilon) chain-specific antisense oligonucleotides (AOs) studied on human myeloma cell line U266 and peripheral blood mononuclear cells of a patient with hypereosinophilia.

Based on cDNA sequence data epsilon chain-specific antisense oligonucleotides were synthesized and checked on in vitro IgE production. Using peripheral blood cells from a hypereosinophilic patient and a human IgE myeloma cell line, U266, marked reduction of in vitro IgE production measured by PRIST was observed. The effect of epsilon antisenses proved to be isotype specific since IgG production by both peripheral blood cells and a lymphoma cell line, CESS, was not affected. Moreover, the expression of other markers on U266 (interleukin-6 receptor and gp130) were not influenced by epsilon-specific antisense oligonucleotides.

Humans↗

PubMed1996

The unusual X-form DNA in oligodeoxynucleotides: dependence of stability on the base sequence and length.

X-form is an unusual double helix of DNA adopted by poly(dA-dT) or (dT-dA)4 at high concentrations of CsF. On the other hand, poly(dA), poly(dT), (dA-dT)4 and most other DNAs do not adopt this conformer. Here we demonstrate that the X-form is strongly destabilized by GC pairs or even minute perturbations of the alternating pyrimidinepurine sequence. For example, the 30-mer d(TATAAT)5, containing five tandem repeats of the Pribnow box, fails to isomerize into the X-form. After (dT-dA)4, the 16-mer (dT-dA)8 is shown to be the second most predisposed oligodeoxynucleotide in the (dT-dA)n series to isomerize into the X-form while the duplex lengths corresponding to n = 3,5,6,7,9,12 and 20 make the X-form unstable even in the strictly alternating (dT-dA)n sequence. Consequently, the (dT-dA)n duplex length is also a crucial factor of the X-form stability on the oligodeoxynucleotide level. We discuss a possibility that the X-form is a solution counterpart of the D-form adopted in dehydrated poly(dA-dT) fibers because properties of these two conformers are remarkably similar in many respects.

Base Sequence↗

PubMed1996

Unusual contribution of 2-aminoadenine to the thermostability of DNA.

The poly(dA-dU) and poly(dI-dC) duplexes have very similar thermostabilities (Tm). This similarity extends also to the pyrimidine 5-methyl group-containing poly(dA-dT) and poly(dI-m5dC). The differences between chemical structures of the A:U and I:C or the A:T and I:m5C base-pairs seem to be unimportant for the thermostability of the DNA. However, on the insertion of an amino group into position 2 of the purines the similarities disappear. Thermostabilities of poly(n2dA-dU) and poly(dG-dC) as well as the poly(n2dA-dT) and poly(dG-m5dC) are radically different. This is also the case with their other 5-substituted pyrimidine-containing derivatives, the 5-ethyl, 5-n-butyl and 5-bromo analogues. The G:C-based polynucleotides are more stable by an average of 40 degrees C than the n2A.U-based ones. Poly(dA,n2dA-dT)-s containing various proportions of A and n2A as well as the natural DNA of S-2L cyanophage that contains n2A bases instead of A were also studied. It was found that dependence of Tm on the n2A-content was non-linear and that the lower Tm is not the consequence of a particular nucleotide sequence. The possible structural reasons for the lower thermostabilization of these B-DNAs by the n2A:T base-pair as compared to the G:C are discussed.

2-Aminopurine↗

PubMed1995

Acyclic oligonucleotide analogues.

Acyclic analogues of oligothymidylate and oligoadenylate and their alternating copolymers were synthesized to study their thermal melting, their stability against snake venom phosphodiesterase and their primer/template properties using the Klenow fragment of the Escherichia coli DNA polymerase I enzyme. Acyclic dodecaadenylate (GlyA)12 hybridized to dodecathymidylate p(dT)12, and the complex presented a sharp melting with a Tm at 24 degrees C. This association was confirmed by circular dichroism curves which were similar to those of the natural oligonucleotide duplexes in A-conformation. (GlyA)12 proved very stable against snake venom phosphodiesterase hydrolysis. The reaction rate was more than 10,000 times slower than that of p(dT)12. (GlyA)12 served as a primer for the Klenow DNA polymerase. When (GlyA)12 was complexed with the poly(dT) template, the enzyme polymerized dATP but the reaction was much slower than with the (GlyT)12 primer. Molecular modelling of atactic (GlyA)12.(dT)12 of the A-conformation indicates that this conformation is energetically possible.

Adenine↗

PubMed1994

Thymine methyl groups stabilize the putative A-form of the synthetic DNA poly(amino2dA-dT).

Poly(amino2dA-dT) easily isomerizes into a non-B conformer which most authors think is an A-form. We synthesized new DNA analogs poly(amino2dA-ethyl5dU) and poly(amino2dA-dU) to show that they do not prefer this conformer. Hence the putative A-form is, like Z-DNA of poly(dG-dC) but unlike A-DNA, strongly stabilized by the methyl group in position 5 of the pyrimidine base. In addition, the putative A-form is induced by divalent cations while it does not need any alcohol to be stable, both properties being typical for Z-DNA again but quite unusual with A-DNA. Despite these similarities, the putative A-form is also distinct from Z-DNA, as poly(amino2dA-dT) is shown to isomerize into a Z-form in the NaCl + NiCl2 solvent system like poly(dA-dT). The present data indicate that the putative A-form of poly(amino2dA-dT) differs in a significant way from all canonical conformers of DNA. Furthermore, the studies of the poly(amino2dA-dT) family of polydeoxynucleotides reveal a novel type of conformational switch in DNA. We also report the B-Z transitions of poly(amino2dA-ethyl5dU) and poly(amino2dA-dU) and their transitions into the putative A-form in aqueous alcohol solutions.

Circular Dichroism↗

PubMed1994

Probing conformational isomerizations of double-stranded poly(dA-dT) by a substitution of minor amounts of the thymine methyls with bulky hydrophobic isopropyl groups.

We probed conformational polymorphism of a synthetic DNA poly(dA-dT) by introducing various small amounts of bulky spherical hydrophobic isopropyl groups into the polynucleotide primary structure. For this purpose, three mixed copolymers of poly(dA-dT,ip5dU) were synthesized in which 2.6%, 8.6% or 14.2% of the polynucleotide pyrimidine bases had the isopropyl group in position 5. The isopropyls made the formation of both A-form and X-form incomplete, and this effect increased with the increasing isopropyl amount in the polynucleotide. However, the polynucleotide isomerization into the A-form was hindered by the isopropyls while the isomerization into the X-form was rather promoted. This observation indicates that, unlike the A-form, the X-form has the base pairs shifted towards the double helix major groove. Z-form was also promoted by the lowest concentration of the isopropyl groups while the most isopropylated poly(dA-dT) aggregated under the Z-form inducing conditions.

Circular Dichroism↗

PubMed1994

UV light-induced crosslinking of the strands of poly(dA-dT) and related alternating purine-pyrimidine DNAs.

Alkaline agarose gel electrophoresis was used to detect UV-induced crosslinking of the strands of poly(dA-dT) and related alternating purine-pyrimidine DNAs in solutions stabilizing various polynucleotide conformations. Strands of the B-form and A-form of poly(dA-dT) were not crosslinked but a UV dose-dependent retarded species appeared in the denaturing gels in parallel with the polynucleotide isomerization into the unusual X-form. Most other polynucleotides adopting the X-form were crosslinked as well. The exceptions include the X-forms of poly(dA-butyl5dU) and poly(dA-pentyl5dU) whose strands do not crosslink because the long exocyclic substituents attached to uracil make the photodimerization impossible. Strands of poly(amino2dA-dT) and poly(dA, amino2dA-dT), the latter polynucleotide containing roughly equal amounts of amino2adenine and adenine, also do not crosslink upon UV irradiation because they isomerize into an A-like conformation which is different from the X-form of poly (dA-dT). In contrast, strands of the mixed copolymers of poly(dA, amino2dA-dT) containing low amino2adenine contents are crosslinked upon UV irradiation, in accordance with the observation that they isomerize into the X-form.

Electrophoresis, Agar Gel↗

PubMed1993

An A-form of poly(amino2dA-dT).poly(amino2dA-dT) induced by polyamines.

The effect of the naturally occurring polyamines spermidine and spermine on poly(amino2-dA-dT).poly(amino2dA-dT) conformation has been studied by UV, CD, and IR spectroscopies. It is shown that a conformational transition is induced in poly(amino2dA-dT).poly(amino2dA-dT) by micromolar concentrations of the polyamines (30 microM) in low-salt aqueous solution. The analysis of our results, in view of previously published studies on conformational properties of the amino polynucleotide, indicates the resulting conformer to be an A-form. Interestingly, the polyamine concentration at the midpoint of the transition is the same in both cases. This provides further evidence that the coordination of positively charged counterions to DNA is determined largely from the DNA structure, probably with an important role for the sequence, and less from the nature of the ions.

Circular Dichroism↗

PubMed1993

Some biochemical properties of an acyclic oligonucleotide analogue. A plausible ancestor of the DNA?

As acyclic oligonucleotides have been suggested as a primitive model of DNA or RNA in prebiotic times, we compared some biochemical properties of these analogues to that of natural ones. Firstly, an acyclic analogue of deoxyribonucleoside triphosphates was tested as a potential substrate of enzymes intervening in nucleic acids synthesis. GlyTTP, a dTTP analogue with a missing 2'-methylene group is not accepted as a substrate by either DNA polymerase or deoxynucleotidyl terminal transferase (TdT). Secondly, the modified dodecathymidylate (GlyT)12, the racemic acyclic sugar analogue of (dT)12, proved to be an efficient primer for DNA polymerase and TdT, though the associative properties of (GlyT)12 are very weak as shown by UV spectroscopy in phosphate buffer without magnesium chloride. But (GlyT)12 has the advantage to be 500-times more stable against hydrolysis by snake venom phosphodiesterase than the corresponding oligothymidylate.

DNA-Directed DNA Polymerase↗