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1.
We report here kinetic and thermodynamic studies on differential isomerization of intramolecular Pyr*Pur.Pyr triplexes in supercoiled plasmids. Two structural isomers of the triplex exist: one with the 3'-half of the Pyr strand as the third strand (H-y3 form) and the other with the 5'-half as the third strand (H-y5 form). The relative populations of the two triplex isomers was determined using the chemical probe with diethyl pryrocarbonate as a function of incubation time. The results demonstrated that triplexes were formed rapidly after a pH change from pH 8.0 to 5.0 and that the initial population of the two isomers exponentially changed with incubation time to reach true thermodynamic equilibrium with a time constant of 0.6-10 h, depending on temperature and the presence of Mg2+. The results clearly demonstrated that interconversion occurs between the two isomers and that the presence of Mg2+ generally retarded the interconversion rates. Kinetic and thermodynamic analyses of the relative populations of the two isomers revealed that the apparent energy barrier for transition from duplex to the H-y3 form is higher than that to the H-y5 form, but H-y3 is more stable in enthalpy terms than H-y5. Therefore, H-y3 is kinetically inferior but thermodynamically favored at higher supercoil levels in plasmids. The presence of Mg2+ resulted in both a kinetic and a thermodynamic preference for H-y5 formation, relative to the H-y3 form. 相似文献
2.
3.
Poly(pyrimidine) . poly(purine) synthetic DNAs containing 5-methylcytosine form stable triplexes at neutral pH 总被引:37,自引:29,他引:8
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Poly(pyrimidine) . poly(purine) tracts have been discovered in the 5'-flanking regions of many eucaryotic genes. They may be involved in the regulation of expression since they can be mapped to the nuclease-sensitive sites of active chromatin. We have found that poly(pyrimidine) . poly(purine) DNAs which contain 5-methylcytosine (e.g. poly[d(Tm5C)] . poly[d(GA)]) will form a triplex at a pH below 8. In contrast, the unmethylated analogue, poly[d(TC)] . poly[d(GA)] only forms a triplex at pHs below 6. Synthetic DNAs containing repeating trinucleotides and poly[d(Um5C)] . poly[d(GA)] behave in a similar manner. Thus the stability of a triplex can be controlled by methylation of cytosine. This suggests a model for the regulation of expression based upon specific triplex formation on the 5'-side of eucaryotic genes. 相似文献
4.
NMR studies of triple-strand formation from the homopurine-homopyrimidine deoxyribonucleotides d(GA)4 and d(TC)4 总被引:18,自引:0,他引:18
The complexes formed by the homopurine and homopyrimidine deoxyribonucleotides d(GA)4 and d(TC)4 have been investigated by one- and two-dimensional 1H NMR. Under appropriate conditions [low pH, excess d(TC)4 strand] the oligonucleotides form a triplex containing one d(GA)4 and two d(TC)4 strands. The homopurine and one of the homopyrimidine strands are Watson-Crick base paired, and the second homopyrimidine strand is Hoogsteen base paired in the major groove to the d(GA)4 strand. Hoogsteen base pairing in GC base pairs requires hemiprotonation of C; we report direct observation of the C+ imino proton in these base pairs. Both homopyrimidine strands have C3'-endo sugar conformations, but the purine strand does not. The major triplex formed appears to have four TAT and three CGC+ triplets formed by binding of the second d(TC)4 strand parallel to the d(GA)4 strand with a 3' dangling end. In addition to the triplexes formed, at least one other heterocomplex is observed under some conditions. 相似文献
5.
Kaushik S Kaushik M Svinarchuk F Malvy C Fermandjian S Kukreti S 《Biochemistry》2011,50(19):4132-4142
Modulation of endogenous gene function, through sequence-specific recognition of double helical DNA via oligonucleotide-directed triplex formation, is a promising approach. Compared to the formation of pyrimidine motif triplexes, which require relatively low pH, purine motif appears to be the most gifted for their stability under physiological conditions. Our previous work has demonstrated formation of magnesium-ion dependent highly stable intermolecular triplexes using a purine third strand of varied lengths, at the purine?pyrimidine (Pu?Py) targets of SIV/HIV-2 (vpx) genes (Svinarchuk, F., Monnot, M., Merle, A., Malvy, C., and Fermandjian, S. (1995) Nucleic Acids Res. 23, 3831-3836). Herein, we show that a designed intramolecular version of the 11-bp core sequence of the said targets, which also constitutes an integral, short, and symmetrical segment (G(2)AG(5)AG(2))?(C(2)TC(5)TC(2)) of human c-jun protooncogene forms a stable triplex, even in the absence of magnesium. The sequence d-C(2)TC(5)TC(2)T(5)G(2)AG(5)AG(2)T(5)G(2)AG(5)AG(2) (I-Pu) folds back twice onto itself to form an intramolecular triple helix via a double hairpin formation. The design ensures that the orientation of the intact third strand is antiparallel with respect to the oligopurine strand of the duplex. The triple helix formation has been revealed by non-denaturating gel assays, UV-thermal denaturation, and circular dichroism (CD) spectroscopy. The monophasic melting curve, recorded in the presence of sodium, represented the dissociation of intramolecular triplex to single strand in one step; however, the addition of magnesium bestowed thermal stability to the triplex. Formation of intramolecular triple helix at neutral pH in sodium, with or without magnesium cations, was also confirmed by gel electrophoresis. The triplex, mediated by sodium alone, destabilizes in the presence of 5'-C(2)TC(5)TC(2)-3', an oligonucleotide complementary to the 3'-oligopurine segments of I-Pu, whereas in the presence of magnesium the triplex remained impervious. CD spectra showed the signatures of triplex structure with A-like DNA conformation. We suggest that the possible formation of pH and magnesium-independent purine-motif triplexes at genomic Pu?Py sequences may be pertinent to gene regulation. 相似文献
6.
Triplex DNA in plasmids and chromosomes 总被引:2,自引:0,他引:2
Circular plasmids containing pyrimidine purine tracts can form both inter-and intramolecular triplexes. Addition of poly(dTC) to plasmid pTC45, which contains a (TC)45.(GA)45 insert, results in intermolecular triplex formation. Agarose-gel electrophoresis gives rise to many well-resolved bands, which correspond to 1, 2, 3, 4... plasmid molecules attached to the added pyrimidine strand. In the electron microscope these complexes appear as a rosette of petals. The mobility of these triplex-containing complexes can be retarded by the addition of a triplex-specific monoclonal antibody, Jel318. Intramolecular triplex formation can be demonstrated at pH 5 in pTC45 and also in pT463-I, a plasmid containing a segment of a crab satellite DNA with both (G)n.(C)n and (TCC)n.(GGA)n inserts. However, although the intermolecular triplex remains stable for some time at pH 8, intramolecular triplex formation only occurs at low pH. Triplexes can also be detected by an immunoblotting procedure with Jel318. This unfamiliar structure is readily demonstrated in eukaryotic extracts, but not in cell extracts from Escherichia coli. Triplexes may thus be an inherent feature of eukaryotic chromosome structure. 相似文献
7.
A structural basis for S1 nuclease sensitivity of double-stranded DNA 总被引:58,自引:0,他引:58
A protonated form of a cloned simple repeating DNA sequence d(TC)n X d(GA) is detectable in equilibrium with the usual Watson-Crick base-paired form at pHs up to 7. This form is anomalously sensitive to a variety of single-strand-specific endonucleases. The observed pH dependent protection of N-7 of dG residues within the insert suggests that these residues are either Hoogsteen or reverse Hoogsteen base-paired to protonated dC residues of the polypyrimidine strand. A structure in which dA:dT Watson-Crick base pairs alternate with Hoogsteen syndG:dCH+ pairs appears to be the most stereochemically acceptable structure consistent with the chemical properties of this protonated DNA. Protonated d(TC)n X d(GA)n interacts with an anti-Z DNA antibody raised against brominated d(GC)n X d(GC)n. 相似文献
8.
Stabilization of PyPuPu triplexes with bivalent cations. 总被引:3,自引:0,他引:3
M D Frank-Kamenetskii V A Malkov O N Voloshin V N Soyfer 《Nucleic acids symposium series》1991,(24):159-162
We studied the formation of stable PyPuPu intermolecular triplexes under neutral pH in the presence of bivalent cations (Mg, Ca, Mn, Co, Ni, Cu, Zn, Cd, and Ba) with the help of the photo- and DMS footprinting assays. The cations which stabilize d(C)n.d(G)n.d(G)n and d(TC)n.d(GA)n.d(AG)n triplexes were determined. Among them, Zn++ ions stabilized both triplexes, whereas Mg++ ions stabilize CGG triplexes, but do not stabilize TC.GA.AG triplexes. We have shown that an arbitrary purine sequence forms the PyPuPu triplex in the presence of Zn++ ions, and that the purine third strand is antiparallel with respect to the purine strand within the duplex. 相似文献
9.
V A Malkov O N Voloshin A G Veselkov V M Rostapshov I Jansen V N Soyfer M D Frank-Kamenetskii 《Nucleic acids research》1993,21(1):105-111
We have studied a protonated pyrimidine-purine-purine (Py-Pu-Pu) triplex, which is formed between the d(C)nd(G)n duplex and the d(AG)m oligonucleotide as the third strand and carries the CG*A+ protonated base-triads. We have observed such an intermolecular complex between a plasmid carrying the d(C)18 d(G)18 insert and the d(AG)5 oligonucleotide without bivalent cations in 200 mM of Na+ at pH4.0. Bivalent cations additionally stabilize the complex. We propose the structures for nearly isomorphous base-triads TA*A, CG*G and CG*A+. To identify the H-DNA-like structure, which includes the triplex between d(C)n d(G)n duplex and the AG-strand, we have cloned in a superhelical plasmid the insert: G10TTAA(AG)5. The data on photofootprinting and chemical modification with diethyl pyrocarbonate, potassium permanganate and dimethyl sulfate demonstrate that the H-like structure with triplex carrying CG*G and CG*A+ base triads is actually formed under acid conditions. In the course of this study we have come across unexpected results on probing of Py-Pu-Pu triplexes by dimethyl sulfate (DMS): the protection effect is observed not only for guanines entering the duplex but also for guanines in the third strand lying in the major groove. We have demonstrated this effect not only for the case the novel protonated Py-Pu-Pu triplex but also for the traditional non-protonated Py-Pu-Pu intramolecular triplex (H*-DNA) formed by the d(C)37 d(G)37 insert in supercoiled plasmid in the presence of Mg2+ ions. 相似文献
10.
Chemical probing of the homopurine.homopyrimidine tract in supercoiled DNA at single-nucleotide resolution 总被引:11,自引:0,他引:11
M Vojtísková S Mirkin V Lyamichev O Voloshin M Frank-Kamenetskii E Palecek 《FEBS letters》1988,234(2):295-299
Local structure of the homopurine.homopyrimidine tract in a supercoiled plasmid pEJ4 was studied using chemical probes at single-nucleotide resolution. The conformation of the homopyrimidine strand was probed by osmium tetroxide, pyridine (Os,py) while that of the homopurine strand was tested by diethyl pyrocarbonate (DEPC), i.e. by probes reacting preferentially with single-stranded DNA. At weakly acidic pH values, a strong Os,py attack on three nucleotides at the centre of the (dC-dT)16 block and a weaker attack on two nucleotides at the end of the block were observed. DEPC modified adenines in the 5'-half of the homopurine strand. Os,py modification at the centre of the block corresponded to the loop of the hairpin formed by the homopyrimidine tract, while DEPC modification corresponded to the unstructured half of the homopurine strand in the model of protonated triplex H form of DNA. 相似文献
11.
The identification of nuclear proteins that bind the homopyrimidine strand of d(GA.TC)n DNA sequences, but not the homopurine strand. 总被引:1,自引:0,他引:1
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I García-Bassets M Ortiz-Lombardía S Pagans A Romero F Canals F X Avil s F Azorín 《Nucleic acids research》1999,27(16):3267-3275
Alternating d(GA.TC)(n)DNA sequences, which are abundant in eukaryotic genomes, can form altered DNA structures. Depending on the environmental conditions, the formation of (GA.GA) hairpins or [C+T(GA.TC)] and [GA(GA.TC)] intramolecular triplexes was observed in vitro. In vivo, the formation of these non-B-DNA structures would likely require the contribution of specific stabilizing factors. Here, we show that Friend's nuclear extracts are rich in proteins which bind the pyrimidine d(TC)(n)strand but not the purine d(GA)n strand (NOGA proteins). Upon chromatographic fractionation, four major proteins were detected (NOGA1-4) that have been purified and characterized. Purified NOGAs bind single-stranded d(TC)n with high affinity and specificity, showing no significant affinity for either d(GA)n or d(GA.TC)nDNA sequences. We also show that NOGA1, -2 and -3, which constitute the three most abundant and specific NOGA proteins, correspond to the single-stranded nucleic acid binding proteins hnRNP-L, -K and -I, respectively. These results are discussed in the context of the possible contribution of the NOGA proteins to the stabilization of the (GA.GA) and [GA(GA.TC)] conformers of the d(GA.TC)n DNA sequences. 相似文献
12.
M Mills P B Arimondo L Lacroix T Garestier C Hélène H Klump J L Mergny 《Journal of molecular biology》1999,291(5):1035-1054
DNA triple helices offer exciting new perspectives toward oligonucleotide-directed inhibition of gene expression. Purine and GT triplexes appear to be the most promising motifs for stable binding under physiological conditions compared to the pyrimidine motif, which forms at relatively low pH. There are, however, very little data available for comparison of the relative stabilities of the different classes of triplexes under identical conditions. We, therefore, designed a model system which allowed us to set up a competition between the oligonucleotides of the purine and pyrimidine motifs targeting the same Watson-Crick duplex. Several conclusions may be drawn: (i) a weak hypochromism at 260 nm is associated with purine triplex formation; (ii) delta H degree of GA, GT and TC triplex formation (at pH 7.0) was calculated as -0.1, -2.5 and -6.1 kcal/mol per base triplet, respectively. This unexpectedly low delta H degree for the purine triple helix formation implies that its delta G degree is nearly temperature-independent and it explains why these triplexes may still be observed at high temperatures. In contrast, the pyrimidine triplex is strongly favoured at lower temperatures; (iii) as a consequence, in a system where two third-strands compete for triplex formation, displacement of the GA or GT strand by a pyrimidine strand may be observed at neutral pH upon lowering the temperature. This original purine-to-pyrimidine triplex conversion shows a significant hypochromism at 260 nm and a hyperchromism at 295 nm which is similar to the duplex-to-triplex conversion in the pyrimidine motif. Further evidence for this triplex-to-triplex conversion is provided by mung bean-nuclease foot-printing assay. 相似文献
13.
Formation of DNA triple helices inhibits DNA unwinding by the SV40 large T-antigen helicase.
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Previous studies have indicated that d(TC)n.d(GA)n microsatellites may serve as arrest signals for mammalian DNA replication through the ability of such sequences to form DNA triple helices and thereby inhibit replication enzymes. To further test this hypothesis, we examined the ability of d(TC)i.d(GA)i.d(TC)i triplexes to inhibit DNA unwinding in vitro by a model eukaryotic DNA helicase, the SV40 large T-antigen. DNA substrates that were able to form triplexes, and non-triplex-forming control substrates, were tested. We found that the presence of DNA triplexes, as assayed by endonuclease S1 and osmium tetroxide footprinting, significantly inhibited DNA unwinding by T-antigen. Strong inhibition was observed not only at acidic pH values, in which the triplexes were most stable, but also at physiological pH values in the range 6.9-7.2. Little or no inhibition was detected at pH 8.7. Based on these results, and on previous studies of DNA polymerases, we suggest that DNA triplexes may form in vivo and cause replication arrest through a dual inhibition of duplex unwinding by DNA helicases and of nascent strand synthesis by DNA polymerases. DNA triplexes also have the potential to inhibit recombination and repair processes in which helicases and polymerases are involved. 相似文献
14.
Parallel-stranded DNA under topological stress: rearrangement of (dA)15.(dT)15 to a d(A.A.T)n triplex. 总被引:1,自引:1,他引:0
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DNA oligonucleotides with appropriate sequences can form a stable duplex in which the two strands are paired in a parallel orientation instead of as the conventional antiparallel double helix of B-DNA. In parallel-stranded DNA (ps-DNA) base pairing is noncanonical with the glycosidic bonds in a trans orientation. The two grooves are equivalent. We have synthesized DNA duplexes consisting of a central parallel-stranded (dA)15.(dT)15 tract flanked by normal antiparallel regions, and ligated them into the pUC18 plasmid. The effect of negative supercoiling on the covalently closed circular molecules was studied by two-dimensional agarose gel electrophoresis and by chemical modification with OsO4-pyridine (Os,py) and diethylpyrocarbonate (DEPC). The following results were obtained: (i) The ps insert, and by inference ps-DNA in general, adopts a right handed helical form. (ii) Upon increasing the negative superhelix density (-sigma) to greater than 0.03 the 15 bp ps insert undergoes a major transition leading to a relaxation corresponding to a reduction in twist of approximately 2.5 helical turns. The transition free surgery is approximately kcal/mol. (iii) The chemical modification pattern of the resulting structure suggests that the purine strand folds back and associates with the pyrimidine strand, forming a novel intramolecular triplex structure consisting of d(A.A.T) base triplets. A model for the triplex conformation is proposed and its thermodynamic properties are analyzed by statistical mechanics. 相似文献
15.
16.
The influence of cations on the capacity of five oligopurine.oligopyrimidine mirror repeat sequences to adopt intramolecular triplexes (the usual H-y3 isomer and/or the rare H-y5 isomer) in recombinant plasmids was investigated. Unexpectedly, the presence of certain metal ions (magnesium, zinc, manganese, and calcium) stabilized the (GAA)4TTCGC(GAA)4 insert in the rare H-y5 when cloned into either of two different sequence backgrounds. Alternatively, either shortening or lengthening the sequence at the central interruption, which becomes the loop of the triplex, led to the formation of the canonical H-y3 under all conditions tested. Similarly, other oligopurine.oligopyrimidine mirror repeat sequences (i.e. (GAA)8 or (GGA)8) formed only the H-y3 under all experimental conditions. All triplexes were stabilized by negative supercoiling at pH 5.0; chemical probe and primer extension analyses served as critical structural tools. Hence, the nature of the central interruption sequence (the loop) of the mirror repeat is important for the stabilization of the H-y5. This region may be important for metal ion binding in the initial stages of triplex formation and thus may play a critical role in determining which isomer is formed. The possible biological role of a DNA sequence adopting three different conformations is discussed. 相似文献
17.
Structure of (dG)n.(dC)n under superhelical stress and acid pH 总被引:7,自引:0,他引:7
V I Lyamichev S M Mirkin M D Frank-Kamenetskii 《Journal of biomolecular structure & dynamics》1987,5(2):275-282
We have recently shown that a (GA)n.(TC)n tract undergoes a sharp structural transition under superhelical stress (V.I. Lyamichev, S.M. Mirkin and M.D. Frank-Kamenetskii, J. Biomol. Struct. Dyn. 2,327 (1985]. Unlike the well studied transitions to the cruciform and to the Z form, this novel transition was strongly pH-dependent. We have found the (dG)n.(dC)n insert to undergo a pH-dependent structural transition similar to that of the (GA)n.(TC)n tract. These new data meet our earlier expectations and disagree with the data of D.E. Pulleyblank, D.B. Haniford and A.R. Morgan, Cell 42, 271 (1985). We conclude that a novel DNA structure (the H-form) is typical of homopurine-homopyrimidine mirror repeats (the H palindromes) under superhelical stress and/or acid pH. In the H-form the homopyrimidine strand forms a hairpin while half of the homopurine strand interacts with the hairpin forming a triplex, the other half of the homopurine strand being unstructured (V.I. Lyamichev, S.M. Mirkin and M.D. Frank-Kamenetskii, J. Biomol. Struct. Dyn. 2, 3, 667 (1986]. 相似文献
18.
Proton nuclear magnetic resonance assignments and structural characterization of an intramolecular DNA triplex. 总被引:3,自引:0,他引:3
Two-dimensional 1H n.m.r. spectroscopy has been used to study the 31-base DNA oligonucleotide 5'-dAGAGAGAACCCCTTCTCTCTTTTTCTCTCTT-3', which folds to form a stable intramolecular triplex in solution at acidic pH. This structure is considerably more difficult to assign than short B-DNA duplexes and requires new assignment methods. The assignment strategy and assignments of almost all of the exchangeable and nonexchangeable resonances are presented. Seven base triplets and one Watson-Crick base-pair form the core of the structure and are connected by a four C and four T loop at either end. The second pyrimidine "strand" (bases 24 to 31) in this intramolecular pyrimidine-purine-pyrimidine triplex binds via Hoogsteen base-pairs in the major groove and is parallel to the purine "strand" (bases 1 to 8). Analysis of the sugar puckers reveals that, contrary to widely accepted belief, the triplex sugars are not predominantly in the N-type (close to C3'-endo) conformation. Except for some of the C nucleotides, all sugars are predominantly S-type (close to C2'-endo). Thus, the duplex DNA does not assume N-type sugar conformations to accommodate a third strand in the major groove. A preliminary model of the triplex structure is presented. 相似文献
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20.
Cation and sequence effects on stability of intermolecular pyrimidine-purine-purine triplex. 总被引:8,自引:7,他引:1
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A differential effect is found of various bivalent cations (Ba2+, Ca2+, Mg2+, Cd2+, Co2+, Mn2+, Ni2+, Zn2+ and Hg2+) on stability of intermolecular Py-Pu-Pu triplex with different sequence of base triads. Ca2+, Mg2+, Cd2+, Co2+, Mn2+, Ni2+ and Zn2+ do stabilize the d(C)n d(G)n d(G)n triplex whereas Ba2+ and Hg2+ do not. Ba2+, Ca2+, Mg2+ and Hg2+ destabilize the d(TC)n d(GA)n d(AG)n triplex whereas Cd2+, Co2+, Mn2+, Ni2+ and Zn2+ stabilize it. The complexes we observe are rather stable because they do not dissociate during time of gel electrophoresis in the co-migration experiments. Chemical probing experiments with dimethyl sulfate as a probe indicate that an arbitrary homopurine-homopyrimidine sequence forms triplex with corresponding purine oligonucleotide in the presence of Mn2+ or Zn2+, but not Mg2+. In the complex the purine oligonucleotide has antiparallel orientation with respect to the purine strand of the duplex. Specifically, we have shown the formation of the Py-Pu-Pu triplex in a fragment of human papilloma virus HPV-16 in the presence of Mn2+. 相似文献