Left-handed Z-DNA and intramolecular triplex formation at the site of an unequal sister chromatid exchange

An unequal sister chromatid exchange (USCE) in the mouse myeloma cell line MPC-11 between 3' regions of the C gamma 2a and C gamma 2b heavy chain genes results in duplication of the C gamma 2a heavy chain gene and generation of a novel recombination joint. The USCE occurs between (TC)n tracts adjacent to alternating purine-pyrimidine tracts. We have investigated the capacity of both the donor regions and the recombinant product involved in this event to adopt left-handed Z-DNA and intramolecular triplexes. The results of chemical probing with diethylpyrocarbonate and osmium tetroxide at the base pair level demonstrate that under the influence of negative supercoiling the alternating purine-pyrimidine regions of these plasmids can adopt Z-DNA at neutral pH, and the oligopurine.oligopyrimidine (pur.pyr) regions of these regions can adopt intramolecular triplexes at low pH (less than or equal to pH 6.0). At intermediate pH values, mixtures of both structures are present. Increasing the negative superhelical density of the plasmid does not increase the amount of triplex present at neutral pH indicating that the presence of long Z-DNA segments adjacent to pur.pyr tract prevents intramolecular triplex formation. In summary, we conclude that the sequences involved in the USCE can form either an intramolecular triplex in the (TC)n tract or Z-DNA in the alternating purine-pyrimidine tract and that Z-DNA will predominate under physiological conditions. The presence of segments which adopt Z-DNA at a site of USCE suggests that formation of this structure may enhance recombination between adjacent pur.pyr tracts.     

Protonated pyrimidine-purine-purine triplex.

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.     

Intramolecular DNA triplexes in supercoiled plasmids. II. Effect of base composition and noncentral interruptions on formation and stability

The effects of interruptions in the homopurine bias and the G+C content of the homopurine.homopyrimidine (pur.pyr) sequences on intramolecular triplex formation and stability in supercoiled plasmids were evaluated. In addition, the interconversion of triplex and duplex, after altering the stabilizing factors (low pH or supercoiling), was studied. We conclude: (a) a 42-base pair pur.pyr sequence with three consecutive interruptions does not form a large triplex with three unpaired nucleotides in the stem. Instead, a mixture of two smaller (27- and 28-nucleotide) triplexes forms. (b) A 28-nucleotide sequence with a single interruption forms a triplex with one unpaired nucleotide in the stem. This interruption causes the triplex to be 7 degrees C less thermostable and requires more superhelical energy for formation than the control triplex. (c) As the G+C content of a pur.pyr sequence increases, the thermostability of the triplex increases and the triplex requires less supercoiling for formation. (d) The interconversion between duplex and triplex is fast. After negative supercoiling is removed, all triplex becomes duplex in about 3 min. When the pH is shifted from 8.0 to 5.2, the conversion of duplex to triplex in a negatively supercoiled plasmid is complete in less than 2 min. Hence, these kinetic properties are consistent with important biological roles for triplexes. In summary, the results from both this and the accompanying paper show that a substantial amount of sequence imperfections is tolerated for triplex formation and stability.     

Detection of triple-helix related structures adopted by poly(dG)-poly(dC) sequences in supercoiled plasmid DNA.

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.     

Central non-Pur.Pyr sequences in oligo(dG.dC) tracts and metal ions influence the formation of intramolecular DNA triplex isomers.

The effect of the central non-Pur.Pyr sequences in oligo(dG.dC) inserts on determining the type of intramolecular DNA triplex isomers formed in negatively supercoiled plasmids was investigated. Different triplex types (H-r3, H-r5, and H-y3), revealed by a combination of chemical probing and Maxam-Gilbert sequencing reactions, were adopted by the oligo(dG.dC) tracts depending on the length and composition of the central non-Pur.Pyr sequences (0, 3, or 5 base pairs) and the kind of metal ions. The H-r3 triplex conformer, one isomer of a Pur.Pur.Pyr structure, was formed in the (C)20 and (C)10GCG(C)10 inserts in plasmids in the presence of certain metal ions. Interestingly, H-r5, the other isomer of the Pur.Pur-Pyr triplex which had not been detected previously, was formed in a (C)9GAATT(C)9 insert in the presence of either Mg2+ or Ca2+. Alternatively, H-y3, one isomer of a Pyr.Pur.Pyr triplex, was formed in the (C)9GAATT(C)9 insert in the absence of metal ions. Thus, central non-Pur.Pyr sequences and metal ions play a role as determinants of the types of intramolecular triplexes formed; they also reduce the requirement of longer Pur.Pyr repeat sequences to form intramolecular triplexes. Furthermore, the effects of MgCl2 concentration and pH on the formation of triplex isomers were examined. The Pur.Pur.Pyr conformations (H-r3 and H-r5) may be the favored conformations in the cellular milieu, since they are stable at physiological pH and metal ion concentration.     

Isolation and characterization of Z-DNA binding proteins from wheat germ

The preparation of a heterogeneous non-histone protein extract from wheat germ utilizing Br-poly(dG-dC).poly(dG-dC) (Z-DNA) affinity chromatography is described. The binding characteristics of antibodies against Z-DNA are used as a model system to define important criteria that the DNA binding behavior of a Z-DNA binding protein should display. We show that the wheat germ extract contains DNA binding proteins specific for left-handed Z-DNA by these criteria. The affinity of the proteins measured by competition experiments was approximately 10(5) greater for Br-poly(dG-dC).poly(dG-dC) (Z-DNA) than for poly(dG-dC).poly(dG-dC) (B-DNA). The affinity of the proteins for plasmid DNA increases with increasing negative superhelicity which is known to stabilize Z-DNA. The proteins are shown to compete with Z-DNA antibodies for binding to supercoiled plasmids. Finally, the affinity for two plasmids at a given superhelical density is greater for the plasmid containing an insert known to form Z-DNA than for a plasmid without the insert. The proteins exhibit a 2-3-fold greater affinity for stretches of (dC-dA)n.(dT-dG)n over stretches of (dG-dC)n.(dG-dC)n when both sequences are induced to form Z-DNA by supercoiling.     

Effect of length, supercoiling, and pH on intramolecular triplex formation. Multiple conformers at pur.pyr mirror repeats

The conformations adopted by five oligopurine.oligopyrimidine (pur.pyr) inserts of various lengths and sequence repeats in recombinant plasmids were evaluated as a function of pH and negative super-helicaldensity. Patterns of chemical reactivity (OsO4 and diethylpyrocarbonate) indicate that long (greater than 36 base pairs) pur.pyr segments can adopt intramolecular triplexes and that increasing the length of the pur.pyr tract reduces the dependence on low pH for structure formation, such that (GA)37 adopts an intramolecular triplex under moderate levels of negative superhelical stress (-sigma = 0.049) at neutral pH. This demonstrates that long pur.pyr segments, which are abundant in eukaryotic genomes, have the potential to adopt triplexes in vivo. Two-dimensional gel electrophoresis of the plasmids combined with chemical probing indicates that for longer sequences, multiple conformers of the intramolecular triplex exist at low pH. These conformers result from nucleation at various positions on the polypurine stretch, giving rise to different extents of relaxation at the same linking number. In addition, the metal ions Co2+, Mn2+, and Mg2+ have profound effects on the pattern of chemical reactivity displayed by long pur.pyr segments at both neutral and low pH, indicating that quite different structures may form in the presence of divalent metal ions. Thus, the types and extent of unusual structures adopted by long pur.pyr segments are complex and heterogeneous, and are dependent on pH, supercoiling, and the presence of divalent cations.     

Intramolecular DNA triplexes in supercoiled plasmids. I. Effect of loop size on formation and stability

The capacity of four oligopurine.oligopyrimidine (pur.pyr) sequences with different lengths of interruptions in the center [GAA)4(N)n(GAA)4G) (n = 3, 5, 7, and 9) to adopt intramolecular DNA triplexes was evaluated in recombinant plasmids. The hyperreactive patterns of the pur.pyr inserts to specific chemical probes (OsO4, diethyl pyrocarbonate, and dimethyl sulfate) at the base pair level demonstrate that intramolecular triplexes with identical 12-base triads in the stem but with different loop sizes (4, 6, 8, and 10 bases) can form in supercoiled plasmids. Furthermore, the extent of OsO4 modification was measured as a function of temperature and of average negative supercoil density. In addition, the transition free energy of B-DNA to triplexes at pH 4.5 was determined by two-dimensional electrophoresis. These comparative studies show that longer loops require more supercoil energy for triplex formation and are less thermostable than triplexes with shorter loops. Also, it may be that not only the loop size but the base composition of the loop region affects the structural transition and triplex stability. Thus, these results significantly broaden the range of natural pur.pyr sequences that may adopt triplexes.     

Nodule DNA in the (GA)37.(CT)37 insert in superhelical plasmids.

Di- or trivalent metal ions stabilize a supercoil-dependent transition in pGA37, which contains the (GA)37.(CT)37 insert, at neutral and basic pH. The structure formed is different from the well known protonated triplexes (H-DNA) adopted at low pH by polypurine.polypyrimidine (Pur.Pyr) inserts in plasmids. DNA samples must be preincubated in the presence of multivalent ions at 50 degrees C for the new transition to occur. At neutral pH in the presence of Co hexamine, both strands of the insert have modification maxima situated at one-third of the distance from both ends. We propose the formation of a new structure called nodule DNA which consists of both Pyr.Pur.Pyr and Pur.Pur.Pyr triplexes and does not contain continuous single-stranded regions. At basic pH (greater than 8.5) in the presence of magnesium ions, the modification pattern corresponds to Pur.Pur.Pyr triplex formation in the whole insert. At neutral pH in the presence of magnesium, both nodule DNA and the Pur.Pur.Pyr triplex can be formed in the insert. We also observed a magnesium-dependent transition at neutral pH in the other Pur.Pyr insert containing plasmids. These data demonstrate that Pur.Pyr sequences can adopt several non-B conformations at close to in vivo conditions.     

Interactions of Drosophila DNA topoisomerase II with left-handed Z-DNA in supercoiled minicircles.

The native form of Drosophila melanogaster DNA topoisomerase II was purified from Schneider's S3 tissue culture cells and studied with two supercoiled minicircle preparations, mini and mini-CG, 354 bp and 370 bp in length, respectively. Mini-CG contains a d(CG)7 insert which assumes a left-handed Z-DNA conformation in negative supercoiled topoisomers with a negative linking number difference - delta Lk greater than or equal to 2. The interactions of topoisomerase II with topoisomer families of mini and mini-CG were studied by band-shift gel electrophoresis in which the individual topoisomers and their discrete or aggregated protein complexes were resolved. A monoclonal anti-Z-DNA IgG antibody (23B6) bound and aggregated only mini-CG, thereby confirming the presence of Z-DNA. Topoisomerase II bound and relaxed mini-CG more readily than mini. In both cases, there was a preference for more highly negatively supercoiled topoisomers. The topoisomerase II inhibitor VM-26 induced the formation of stable covalent DNA-protein intermediates. In addition, the non-hydrolyzable GTP analogue GTP gamma S inhibited the binding and relaxation activities. Experiments to detect topoisomerase cleavage sites failed to elicit specific loci on either minicircle preparation. We conclude that Drosophila topoisomerase II is able to bind and process small minicircles with lengths as short as 360 bp and negative superhelix densities, - sigma, which can exceed 0.1. Furthermore, the enzyme has a preferential affinity for topoisomers containing Z-DNA segments and relaxes these molecules, presumably by cleavage external to the inserts. Thus, a potentially functional relationship between topoisomerase II, an enzyme regulating the topological state of DNA-chromatin in vivo, and left-handed Z-DNA, a conformation stabilized by negative supercoiling, has been established.     

Triplex DNA in plasmids and chromosomes

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.     

DNA structural transitions within the PKD1 gene.

Autosomal dominant polycystic kidney disease (ADPKD) affects over 500 000 Americans. Eighty-five percent of these patients have mutations in the PKD1 gene. The focal nature of cyst formation has recently been attributed to innate instability in the PKD1 gene. Intron 21 of this gene contains the largest polypurine. polypyrimidine tract (2.5 kb) identified to date in the human genome. Polypurine.polypyrimidine mirror repeats form intramolecular triplexes, which may predispose the gene to mutagenesis. A recombinant plasmid containing the entire PKD1 intron 21 was analyzed by two-dimensional gel electrophoresis and it exhibited sharp structural transitions under conditions of negative supercoiling and acidic pH. The superhelical density at which the transition occurred was linearly related to pH, consistent with formation of protonated DNA structures. P1 nuclease mapping studies of a plasmid containing the entire intron 21 identified four single-stranded regions where structural transitions occurred at low superhelical densities. Two-dimensional gel electrophoresis and chemical modification studies of the plasmid containing a 46 bp mirror repeat from one of the four regions demonstrated the formation of an H-y3 triplex structure. In summary, these experiments demonstrate that a 2500 bp polypurine.polypyrimidine tract within the PKD1 gene is capable of forming multiple non-B-DNA structures.     

Presence of divalent cation is not mandatory for the formation of intramolecular purine-motif triplex containing human c-jun protooncogene target

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.     

Cruciform extrusion facilitates intramolecular triplex formation between distal oligopurine.oligopyrimidine tracts: long range effects.

Two short d(AG)n tracts separated by either of two 40-base pair (bp) inverted repeats adopt a triple-stranded conformation (H-DNA) at the bottom of an extruded cruciform stem in negatively supercoiled plasmids at pH 4.5. Plasmids containing one d(AG)n adjacent to the inverted repeat or containing two d(AG)n tracts separated by a random sequence did not form the triplex structures. These conclusions were derived from chemical modification patterns and UV-sensitivity studies. Two-dimensional agarose gel electrophoresis revealed that the entire insert containing the cruciform and the triplex is locally unlinked. Moreover, a long range structural effect (over 40 bp of random sequence) of one (AG)7 block on the behavior of a second (AG)7 sequence was detected. This effect cannot be transmitted throughout a 50-bp segment of random sequence. A GenBank search revealed the frequent occurrence of short oligopurine blocks with an intervening random sequence of 17-40 bp.     

Long (dA)n.(dT)n tracts can form intramolecular triplexes under superhelical stress.

Plasmids containing long tracts of (dA)n.(dT)n have been prepared and their conformations examined in linear and supercoiled DNA using a series of chemical and enzymic probes which are known to be sensitive to unusual DNA structures. Under superhelical stress and in the presence of magnesium the sequence T69.A69 adopts a conformation at pH 8.0 consistent with the formation of an intramolecular DNA triplex. Site specific cleavage of the supercoiled plasmid by single-strand specific nucleases occurs within the A.T insert; the 5'-end of the purine strand is sensitive to reaction with diethylpyrocarbonate while the central 5-6 bases of the pyrimidine strand are reactive to osmium tetroxide. By contrast shorter inserts of A33.T33 and A23.T23 do not appear to form unusual structures.     

Unusual protonated structure in the homopurine.homopyrimidine tract of supercoiled and linearized plasmids recognized by chemical probes

Plasmid pEJ4, which is a derivative of pUC19 containing an insert with 60-bp-long homopurine.homopyrimidine tract from sea urchin P. miliaris histone gene spacer, was studied by chemical probes of the DNA structure osmium tetroxide and glyoxal. The former probe reacts with pyrimidine bases, while the latter forms a stable product only with guanine residues. These probes can thus be applied as specific probes for the homopyrimidine and homopurine strands, respectively. At pH 6.0 the site-specific modification of the homopurine.homopyrimidine tract by both probes was observed at native superhelical density of the plasmid. In the linear plasmid under the same conditions this modification was absent; it appeared, however, at more acid pH values. In supercoiled DNA the hypersensitivity of the homopurine.homopyrimidine tract to osmium tetroxide did not substantially change when pH was decreased from 6.0 to 4.0. Changes in NaCl concentration at pH 4.5 did not influence the hypersensitivity to osmium tetroxide; at pH 6.0 this hypersensitivity decreased with increasing NaCl concentration. These results thus show that the chemical probes recognize an unusual protonated structure containing unpaired bases or non-Watson-Crick base pairs. At pH 5.6 the site-specific modification occurred at or near to the middle of the homopurine.homopyrimidine tract, suggesting that a hairpin may be involved in the unusual structure under the given conditions. From the models suggested so far for the unusual structure of homopurine.homopyrimidine tracts our results fit best the protonated triplex H form suggest by V.I. Lyamichev, S.M. Mirkin and M.D. Frank-Kamenetskii, J. Biomol. Struct. Dyn. 3,667 (1986).     

Polypurine/polypyrimidine sequences with the potential of forming triplexes in the proviral DNA of bovine retroviruses

The perfect interstrand triplexes that could potentially arise in the proviral DNA of two widespread cattle retroviruses such as bovine leukemia virus (BLV) and bovine immunodeficiency virus (BIV) were determined. The fragments, which formed triplexes at acidic pH, were found in the genomes of both viruses; five fragments were found in BVL and 10 fragments in BIV. One of these fragments (it is localized in the BVL gag gene) might exist like a part of a cruciform structure. Existence of the triplexes was experimentally confirmed by visualization of supercoiled pGEMEX DNA with the use of atomic force microscopy; six fragments with mirror symmetry, which are necessary for formation of intramolecular triplexes, were found. Triplexes represent one of the elements of the signaling mechanisms of the genome function. Maps of triplex location in the cattle retroviral genome were built.     

Topoisomerase mutants and physiological conditions control supercoiling and Z-DNA formation in vivo.

The influence of topoisomerase I and gyrase mutations in Escherichia coli on the supercoiled density of recombinant plasmids and the stability of left-handed Z-DNA was investigated. The formation of Z-DNA in vivo by dC-dG sequences of different lengths was used to determine the effective plasmid supercoil densities in the mutant strains. The presence of Z-DNA in the cells was detected by linking number and EcoRI methylase inhibition assays. A change in the unrestrained superhelical tension in vivo directly effects the B- to Z-DNA transition. Alterations in the internal or external environment of the cells, such as the inactivation of gyrase or topoisomerase I, a gyrase temperature-sensitive mutant, or starvation of cells, have a dramatic influence on the topology of plasmids. Also, E. coli has significantly more superhelical strain than Klebsiella, Morganella, or Enterobacter. These studies indicate that linking deficiency and effective supercoil density are mutually independent variables of plasmid tertiary structure. A variety of factors, such as protein-DNA interactions, activity of topoisomerases, and the resulting supercoil density, contribute to the B to Z transition inside living cells.