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1.
Fourier transform infrared (FTIR), UV absorption and exchangeable proton NMR spectroscopies have been used to study the formation and stability of two intramolecular pH-dependent triple helices composed by a chimeric 29mer DNA-RNA (DNA double strand and RNA third strand) or by the analogous 29mer RNA. In both cases decrease of pH induces formation of a triple helical structure containing either rU*dA.dT and rC+*dG.dC or rU*rA.rU and rC+*rG.rC triplets. FTIR spectroscopy shows that exclusively N-type sugars are present in the triple helix formed by the 29mer RNA while both N- and S-type sugars are detected in the case of the chimeric 29mer DNA-RNA triple helix. Triple helix formation with the third strand RNA and the duplex as DNA appears to be associated with the conversion of the duplex part from a B-form secondary structure to one which contains partly A-form sugars. Thermal denaturation experiments followed by UV spectroscopy show that a major stabilization occurs upon formation of the triple helices. Monophasic melting curves indicate a simultaneous disruption of the Hoogsteen and Watson-Crick hydrogen bonds in the intramolecular triplexes when the temperature is increased. This is in agreement with imino proton NMR spectra recorded as a function of temperature. Comparison with experiments concerning intermolecular triplexes of identical base and sugar composition shows the important role played by the two tetrameric loops in the stabilization of the intramolecular triple helices studied.  相似文献   

2.
Negative superhelical strain induces the poly(dG)-poly(dC) sequence to adopt two totally different types of triple-helices, either a dG.dG.dC triplex in the presence of Mg(+)+ at both neutral and acidic pHs or a protonated dC+.dG.dC triplex in the absence of Mg(+)+ ions at acidic pH (1). To examine whether there are still other types of non-B DNA structures formed by the same sequence, we constructed supercoiled plasmid DNAs harboring varying lengths of the poly(dG) tract, and the structures adopted by each supercoiled plasmid DNA were studied with a chemical probe, chloroacetaldehyde. The potential of a poly(dG)-poly(dC) sequence to adopt non-B DNA structures depends critically on the length of the tract. Furthermore, in the presence of Mg(+)+ and at a mildly acidic pH, in addition to the expected dG.dG.dC triplex detected for the poly(dG) tracts of 14 to 30 base pairs (bp), new structures were also detected for the tracts longer than 35 bp. The structure formed by a poly(dG) tract of 45 bp revealed chemical reaction patterns consistent with a dG.dG.dC triplex and protonated dC+.dG.dC triple-helices fused together. This structure lacks single-stranded stretches typical of intramolecular triplexes.  相似文献   

3.
Optical spectroscopic properties of 4',6-diamidino-2-phenylindole (DAPI) and ethidium bromide complexed with poly(dG).poly(dC).poly(dC)(+) triplex and poly(dG).poly(dC) duplex were compared in this study. When complexed with both duplex and triplex, ethidium is characterized by hypochromism and a red shift in the absorption spectrum, a complicate induced circular dichroism (CD) band in the polynucleotide absorption region, and a negative reduced linear dichroism signal in both polynucleotide and drug absorption regions. The spectral properties for both duplex- and triplex-bound ethidium are identical and both can be understood by the intercalation binding mode. In contrast, the absorption and CD spectra of DAPI complexed with triplex differ from those of the DAPI-duplex complex, although both complexes can be understood by the intercalation binding mode. Considering that the third strand runs along the major groove of the template duplex, we conclude that the DAPI molecule partially intercalates near the major groove of the duplex, where the third strand can affect its spectroscopic properties.  相似文献   

4.
The formation of an intramolecular dG.dG.dC triplex in Escherichia coli cells is demonstrated at single-base resolution. The intramolecular dG.dG.dC triplex structure was probed in situ for E. coli cells containing plasmid DNAs with varying lengths of poly(dG).poly(dC) tracts employing chloroacetaldehyde. This chemical probe reacts specifically with unpaired DNA bases. The triplex structure formed with the poly(dG).poly(dC) tracts of 35 and 44 base-pairs, but not with 25 base-pairs. The triplex was detected only one to two hours after the chloramphenicol treatment: the period at which the extracted plasmid DNA revealed the maximal superhelical density.  相似文献   

5.
A study of the interaction between poly(dG)-poly(dC) and poly(rC) demonstrates that, at neutral pH and high ionic strength, there is replacement of the dC strand by poly(rC). At acid pH, formation of a triple-stranded complex which equally may involve the replacement phenomenon is observed. There is no evidence for interaction at neutral pH between poly(dG)-poly(dC) and oligo(rC), while a three-stranded complex is formed at acid pH. These data are consistent with the studies of comparative stabilities of double stranded deoxy or ribo polymers and deoxy-ribo hybrids.  相似文献   

6.
The binding site and the geometry of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (CoTMPyP) complexed with double helical poly(dA).poly(dT) and poly(dG).poly(dC), and with triple helical poly(dA).[poly(dT)](2) and poly(dC).poly(dG).poly(dC)(+) were investigated by circular and linear dichroism (CD and LD). The appearance of monomeric positive CD at a low [porphyrin]/[DNA] ratio and bisignate CD at a high ratio of the CoTMPyP-poly(dA).poly(dT) complex is almost identical with its triplex counterpart. Similarity in the CD spectra was also observed for the CoTMPyP-poly(dG).poly(dC) and -poly(dC).poly(dG).poly(dC)(+) complex. This observation indicates that both monomeric binding and stacking of CoTMPyP to these polynucleotides occur at the minor groove. However, different binding geometry of CoTMPyP, when bind to AT- and GC-rich polynucleotide, was observed by LD spectrum. The difference in the binding geometry may be attributed to the difference in the interaction between polynucleotides and CoTMPyP: in the GC polynucleotide case, amine group protrude into the minor groove while it is not present in the AT polynucleotide.  相似文献   

7.
Hydrogen exchange study of some polynucleotides and transfer RNA   总被引:11,自引:0,他引:11  
The apparent disagreement between published transfer RNA hydrogen exchange results and the tRNA cloverleaf model, prompted a re-investigation of the relationship between hydrogen exchange data and nucleic acid structure. Hydrogen-tritium exchange experiments were carried out with samples of pure and mixed tRNA and with the synthetic polynucleotide bihelices: poly(rA) · poly(rU), poly(rI) · poly(rC), poly(rG) · poly(rC) and poly(dG) · poly (dC).  相似文献   

8.
Duplex DNA containing oligo(dG.dC)-rich clusters can be isolated by specific binding to poly(rC)-Sephadex. This binding, probably mediated by the formation of an oligo(dG.dC)rC+ triple helix, is optimal at pH 5 in 50% formamide, 2 M LiCl; the bound DNA is recovered by elution at pH 7.5. Using this method we find that the viral DNAs PM2, lambda and SV40 contain at least 1, 1 and 2 sites for binding to poly(rC)-Sephadex, respectively. These binding sites have been mapped in the case of SV40; the binding sites can in turn be used for physical mapping studies of DNAs containing (dG.dC) clusters. Inspection of the sequence of the bound fragments of SV40 DNA shows that a (dG.dC)6-7 tract is required for the binding of duplex DNA to poly(rC)-Sephadex. Although about 60% of rabbit DNA cleaved with restriction endonuclease KpnI binds to poly(rC)-Sephadex, no binding is observed for the 5.1 kb DNA fragment generated by KpnI digestion, which contains the rabbit beta-globin gene. This indicates that oligo(dG.dC) clusters are not found close to the rabbit beta-globin gene.  相似文献   

9.
10.
The binding site and the geometry of Co(III)meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (CoTMPyP) complexed with double helical poly(dA)·poly(dT) and poly(dG)·poly(dC), and with triple helical poly(dA)·[poly(dT)]2 and poly(dC)·poly(dG)·poly(dC)+ were investigated by circular and linear dichroism (CD and LD). The appearance of monomeric positive CD at a low [porphyrin]/[DNA] ratio and bisignate CD at a high ratio of the CoTMPyP-poly(dA)·poly(dT) complex is almost identical with its triplex counterpart. Similarity in the CD spectra was also observed for the CoTMPyP-poly(dG)·poly(dC) and -poly(dC)·poly(dG)·poly(dC)+ complex. This observation indicates that both monomeric binding and stacking of CoTMPyP to these polynucleotides occur at the minor groove. However, different binding geometry of CoTMPyP, when bind to AT- and GC-rich polynucleotide, was observed by LD spectrum. The difference in the binding geometry may be attributed to the difference in the interaction between polynucleotides and CoTMPyP: in the GC polynucleotide case, amine group protrude into the minor groove while it is not present in the AT polynucleotide.  相似文献   

11.
In this paper, we describe a production procedure of the one-to-one double helical complex of poly(dG)–poly(dC), characterized by a well-defined length (up to 10 kb) and narrow size distribution of molecules. Direct evidence of strands slippage during poly(dG)–poly(dC) synthesis by Klenow exo fragment of polymerase I is obtained by fluorescence resonance energy transfer (FRET). We show that the polymer extension results in an increase in the separation distance between fluorescent dyes attached to 5′ ends of the strands in time and, as a result, losing communication between the dyes via FRET. Analysis of the products of the early steps of the synthesis by high-performance liquid chromatography and mass spectroscopy suggest that only one nucleotide is added to each of the strand composing poly(dG)–poly(dC) in the elementary step of the polymer extension. We show that proper pairing of a base at the 3′ end of the primer strand with a base in sequence of the template strand is required for initiation of the synthesis. If the 3′ end nucleotide in either poly(dG) or poly(dC) strand is substituted for A, the polymer does not grow. Introduction of the T-nucleotide into the complementary strand to permit pairing with A-nucleotide results in the restoration of the synthesis. The data reported here correspond with a slippage model of replication, which includes the formation of loops on the 3′ ends of both strands composing poly(dG)–poly(dC) and their migration over long-molecular distances (μm) to 5′ ends of the strands.  相似文献   

12.
Sugimoto N  Nakano M  Nakano S 《Biochemistry》2000,39(37):11270-11281
Thermodynamics of 66 RNA/DNA duplexes containing single mismatches were measured by UV melting methods. Stability enhancements for rG. dT mismatches were the largest of all mismatches examined here, while rU.dG mismatches were not as stable. The methyl group on C5 of thymine enhanced the stability by 0.12 approximately 0.53 kcal mol(-)(1) depending on the identity of adjacent Watson-Crick base pairs, whereas the 2'-hydroxyl group in ribouridine stabilized the duplex by approximately 0.6 kcal mol(-)(1) regardless of the adjacent base pairs. Stabilities induced by the methyl group in thymine, the 2'-hydroxyl group of ribouridine, and an nucleotide exchange at rG.dT and rU.dG mismatches were found to be independent of each other. The order for the mismatch stabilities is rG.dT > rU. dG approximately rG.dG > rA.dG approximately rG.dA approximately rA. dC > rA.dA approximately rU.dT approximately rU.dC > rC.dA approximately rC.dT, although the identity of the adjacent base pairs slightly altered the order. The pH dependence stability and structural changes were suggested for the rA.dG but not for rG.dA mismatches. Comparisons of trinucleotide stabilities for G.T and G.U pairs in RNA, DNA, and RNA/DNA duplexes indicate that stable RNA/DNA mismatches exhibit a stability similar to RNA mismatches while unstable RNA/DNA mismatches show a stability similar to that of DNA mismatches. These results would be useful for the design of antisense oligonucleotides.  相似文献   

13.
A Ray  G S Kumar  S Das  M Maiti 《Biochemistry》1999,38(19):6239-6247
The interaction of aristololactam-beta-D-glucoside (ADG), a DNA intercalating alkaloid, with the DNA triplexes, poly(dT). poly(dA)xpoly(dT) and poly(dC).poly(dG)xpoly(dC+), and the RNA triplex poly(rU).poly(rA)xpoly(rU) was investigated by circular dichroic, UV melting profile, spectrophotometric, and spectrofluorimetric techniques. Comparative interaction with the corresponding Watson-Crick duplexes has also been examined under identical experimental conditions. Triplex formation has been confirmed from biphasic thermal melting profiles and analysis of temperature-dependent circular dichroic measurements. The binding of ADG to triplexes and duplexes is characterized by the typical hypochromic and bathochromic effects in the absorption spectrum, quenching of steady-state fluorescence intensity, a decrease in fluorescence quantum yield, an increase or decrease of thermal melting temperatures, and perturbation in the circular dichroic spectrum. Scatchard analysis indicates that ADG binds both to the triplexes and the duplexes in a noncooperative manner. Binding parameters obtained from spectrophotometric measurements are best fit by the neighbor exclusion model. The binding affinity of ADG to the DNA triplexes is substantially stronger than to the RNA triplex. Thermal melting study further indicates that ADG stabilizes the Hoogsteen base-paired third strand of the DNA triplexes whereas it destabilizes the same strand of RNA triplex but stabilizes its Watson-Crick strands. Comparative data reveal that ADG exhibits a stronger binding to the triple helical structures than to the respective double helical structures.  相似文献   

14.
A circular dichroism study of poly dG, poly dC, and poly dG:dC   总被引:22,自引:0,他引:22  
D M Gray 《Biopolymers》1974,13(10):2087-2102
We have measured the ultraviolet circular dichroism spectra of oligo d(pG)5, poly dN AcG, poly dI, poly dC, two samples of poly dG, and four samples containing double-stranded poly dG:dC. We find that oligo d(pG)5 and poly dG exist in self-complexed forms as well as in single-stranded forms. Unlike the self-complexed form of poly dG, the single-stranded form of poly dG can hydrogen-bond with single-stranded poly dC. We present spectral data for double-stranded poly dG:dC, which can be used to help characterize poly dG:dC preparations and which provide a basis for resolving discrepancies among other reported poly dG:dC spectra.  相似文献   

15.
The synthesis of polydeoxyribose polymers by Escherichia coli DNA polymerase I has been investigated with control and gamma-irradiated DNA-like polymer templates containing only two bases. The results show that irradiation of a poly(dA) strand leads to the incorporation of dG, whereas irradiation of poly(dC) and poly(dG) strands both lead to the incorporation of dA. Irradiation of poly(dT) does not lead to the incorporation of any wrong base. The wrong bases are incorporated into the complementary strand of the newly synthesised DNA.  相似文献   

16.
Plasmids containing (dG)27.(dC)27 inserts (pPG27), (dG)37.(dC)37 inserts (pPG37), and (dG)24C(dG)21.(dC)24G(dC)21 inserts (pPG46C) were constructed for the study of structural transitions within (dG)n.(dC)n stretches. Two-dimensional gel electrophoresis has shown that a Mg2+-dependent supercoiling-induced structural transition takes place at pH 8 in plasmid pPG46C. The transition occurs at -0=0.06 and involves a supercoiling release corresponding to 5 superhelical turns. After denaturation of the restriction fragments containing (dG)n.(dC)n inserts, the strands do not renature completely and (dG)n-containing strand migrates in PAGE much faster than the (dC)n-containing one. Chemical modification experiments with the (dG)n-strand have revealed the periodic nature of the protection of guanines against dimethyl sulfate methylation. The (dG)n strand in the presence of Mg2+ forms complexes with the complementary (dC)n strand, which differ from the native duplex in mobility. We believe these effects to be due to the formation of an intrastrand structure within the (dG)n strand stabilized by G.G interactions (we called it G-structure), which in the presence of Mg2+ forms an interstrand complex. with the (dC)n strand.  相似文献   

17.
An endonuclease activity (termed endonuclease G) that selectively cleaves DNA at (dG)n X (dC)n tracts has been partially purified from immature chicken erythrocyte nuclei. Sites where n greater than or equal to 9 are cleaved in a manner that resembles types II and III restriction nucleases. The nicking rate of the G-strand is 4- to 10-fold higher than that of the C-strand depending on the length of the (dG)n X (dC)n tract and/or nucleotide composition of the flanking sequences. Endonuclease G hydrolyzes (dG)24 X (dC)24 of supercoiled DNA in a bimodal way every 9-11 nucleotides, the maxima in one strand corresponding to minima in the opposite, suggesting that it binds preferentially to one side of the double helix. The nuclease produces 5' phosphomonoester ends and its activity is dependent on Mg2+ or Mn2+. The wide distribution and high relative activity of endonuclease G in a variety of tissues and species argues for a general role of the enzyme. The striking correlation between genetic instability and poly(dG) X poly(dC) tracts in DNA suggests that these sequences and endonuclease G are involved in recombination processes.  相似文献   

18.
Alkaline titrations of different samples of poly(dG).poly(dC) and of the constituent homopolymers poly(dG) and poly(dC) have been performed in 0.15 M NaCl and their CD spectra followed. Sample I contained a slight excess of poly(dC) (52% C: 48% G) and showed a single reversible transition (pK = 11.9) due to the dissociation of double stranded poly(dG).poly(dC). Sample II, containing an excess of poly(dG) (43% C: 57% G), showed two transitions (pK1 = 11.4, PK2 = 11.9) the first one being only partially reversible. Examination of the CD spectra along the alkaline titrations indicated the presence of another hydrogen-bonded complex of higher G content. Mixing curves performed at pH 8 have confirmed the presence of a 2G: 1C complex, besides the double stranded complex. It can be formed in amounts up to 30% by mixing the two homopolymers, alkali treatment and heating. The CD spectra of the two complexes have been computed from the CD data of the mixing curves. This permitted the determination of the concentrations of both complexes and homopolymers in all samples. The ratio of triple to double stranded complex is not only dependent on the G/C ratio of the sample, but also a function of the previous physico-chemical conditions. These results explain the variability of many properties of different poly(dG).poly(dC) samples observed by other workers.  相似文献   

19.
Raman spectra of polyribocytidylic acid show the formation of an ordered single-stranded structure [poly(rC)] at neutral pH and an ordered double-stranded structure containing hemiprotonated bases [poly(rC)·poly(rC+)] in the range 5.5 > pH > 3.7. Below 40°C, poly(rC) contains stacked bases and a backbone geometry of the A-type, both of which are gradually eliminated by increasing the temperature to 90°C. Below 80°C, poly(rC)·poly(rC+) contains bases which are hydrogen bonded and stacked and a backbone geometry also of the A-type. In this structure the bases of each strand are shown to be structurally identical, i.e., hemiprotonated, and therefore distinct from both neutral and protonated cytosines. Infrared and Raman spectra indicate the existence of a center of symmetry with respect to the paired cytosine residues, which suggests that the additional proton per base pair is shared equally by the two hydrogen-bonded bases. Denaturation of poly(rC)·poly(rC+) occurs cooperatively (tm ≈ 80°C) with elimination of base stacking, base pairing, and the A-helix geometry. Each of the separated strands of the denatured complex is shown to contain comparable amounts of both neutral and protonated cytosines, most likely in alternating sequence [poly(rC, rC+)]. In both poly(rC, rC+) and poly(rC), at 90°C, the backbones do not exhibit the phosphodiester Raman frequencies characteristic of other disordered polyribonucleotide chains. This is interpreted to mean that the single strands, though devoid of base stacking and A-type structure, contain uniformly ordered backbones of a specific type. Fully protonated poly(rC+), on the other hand, forms no ordered structure and may be characterized as a disordered (random chain) polynucleotide at all temperatures. Several Raman lines of poly(rC) are absent from the spectrum of poly(rC)·poly(rC+) and vice versa. These frequencies, assigned mainly to vibrations of the ribose groups, suggest that the furanose ring conformations are different in the single-stranded and double-stranded structures of polyribocytidylic acid. Several other Raman group frequencies have been identified and correlated with the polymer secondary structures.  相似文献   

20.
Conformation of nucleic acids and the analysis of the hypochromic effect   总被引:8,自引:5,他引:3  
The spectra of two double-helical RNA species isolated from virus-like particles found in the mycelium of Penicillium chrysogenum were measured at about 25 degrees C, and at 95 degrees C after denaturation to a single-stranded form, and compared with the spectrum of the equivalent mixture of nucleotides. For both species the difference spectrum (epsilon(95 degrees C)-epsilon(25 degrees C)) when increased by 33% was very similar to the difference spectrum (epsilon(nucleotides)-epsilon(25 degrees C)). In contrast it was found for rat liver DNA that there was no simple relation between (epsilon(95 degrees C)-epsilon(25 degrees C)) and (epsilon(nucleotides)-epsilon(25 degrees C)). These observations were accounted for on the basis of the difference spectra for rA.rU, rG.rC, dA.dT and dG.dC base-pairs derived from model polyribonucleotides and polydeoxyribonucleotides. The difference spectra (epsilon(95 degrees C)-epsilon(25 degrees C)) found for rRNA and those calculated from a combination of the difference spectra for rA.rU and rG.rC base-pairs were in good agreement.  相似文献   

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