Enhanced reactivity of a B-Z junction for cleavage by the restriction enzyme MboI

We have been investigating the structure, dynamics, and ligand-binding properties of the interface that exists between a right-handed conformation and a left-handed conformation (i.e., a B-Z junction) in synthetic DNA oligomers. Since exo- and endonuclease activity is known to be sensitive to the conformation of the template DNA, we have designed and synthesized a DNA oligonucleotide of 20 base pairs (designated as BZ-III) with an MboI recognition site (GATC) at the location of a potential B-Z junction. The activity of the MboI enzyme toward this molecule and DNA oligomers that contain multiple MboI sites located at B-Z junctions was monitored in the absence and presence of the Z-conformation-inducing reagent cobalt hexaammine. In all cases, the activity of the enzyme was enhanced in the presence of cobalt hexaammine. The activity of MboI toward BZ-III, in the presence and absence of cobalt hexaammine, was also examined when the DNA oligomer is also in the presence of the DNA binding drugs actinomycin D, ametantrone, or ethidium bromide. In all cases, the activity of the enzyme was inhibited in the presence of drug. The results suggest that B-Z junctions are structurally unique and that this uniqueness may alter nuclease activity at sites in or near the junction.     

Anomalous gel migration of DNA oligomers containing multiple conformational junctions

We have previously shown that a short 16 base pair DNA oligomer can accommodate a B-Z conformational junction [Sheardy, R. D., & Winkle, S. A. (1989) Biochemistry 28, 720-725]. Results from 1H NMR studies indicated that only three base pairs were involved in the junction and that one of these base pairs was highly distorted. Being interested in the nature of this distortion, we constructed DNA oligomers which have the potential to contain multiple B-Z junctions for polyacrylamide electrophoretic studies. We report that the mobilities displayed by these molecules through acrylamide gels in the absence and presence of cobalt suggest that these molecules run shorter than they actually are. This anomalous migration may be due to structural/dynamic properties of the DNA helix manifested by the periodic distortions of the potential B-Z junctions.     

Temperature-dependent CD and NMR studies on a synthetic oligonucleotide containing a B-Z junction at high salt

It is now accepted that two or more conformations may exist within the same DNA molecule, thereby generating conformational junctions. The presence of B-Z junctions between right- and left-handed DNA conformations has been detected in plasmids by a number of techniques. Preliminary characterization of the first example of a B-Z junction is a short DNA oligonucleotide has recently been reported [Sheardy, R. D. (1988) Nucleic Acids Res. 16, 1153-1167]. We report additional CD and NMR data that support the existence of the junction in this model oligomer. These studies indicate that only three base pairs are involved in the junction and only one of these is dramatically distorted. Furthermore, the NMR saturation-transfer experiments suggest the junction's internal motion is temperature dependent.     

Polynucleotide block polymers consisting of a DNA.RNA hybrid joined to a DNA.DNA duplex. Synthesis and characterization of dGn.rCidCk duplexes.

The synthesis of several nucleic acid block polymers of the general type dGn.rCidCk is described. The key steps in this procedure were the joining of dCk oligomers, protected at the 3'-OH with an acetyl group, to rCi oligomers by T4 DNA ligase and the purification of the products by RPC-5 column chromatography. The block polymers were characterized by 20% polyacrylamide gel electrophoresis, UV and CD spectra, analytical Cs2SO4 buoyant density analyses, helix-coil transitions and S1 nuclease studies. NMR studies on one member of this series, dGn.rC11dC16, were reported recently (Selsing, E., Wells, R.D., Early, T.A., and Kearns, D.R. (1978) Nature 275, 249-250). The NMR studies and the results described herein indicate that these block polymers are linear duplexes with two adjoining conformations yet are hydrogen-bonded and base-stacked throughout with minimal disruption of the helix at the junction of the two conformations. Computer model building studies described in the following paper (Selsing, E., Wells, R.D., Alden, C.J., and Arnott, S. (1979) J. Biol. Chem. 254, 5417-5422) predict that these nucleic acids contain a bend at the junction region.     

Preliminary spectroscopic characterization of a synthetic DNA oligomer containing a B-Z junction at high salt.

It is well known that the local conformation of a segment of DNA is dependent upon both the sequence of the segment and the conditions under which the DNA is prepared. In extreme cases, the DNA may contain regions of both right and left-handed conformations, mandating the existence of a conformational junction between the two. These B-Z junctions have been observed in plasmids but, to date, no model systems have been characterized to determine the molecular nature of these junctions. Preliminary CD, UV, and NMR studies on such a model are presented here. A 16 base pair oligonucleotide, containing a potential B-Z junction, has been synthesized and characterized by the above techniques. The results suggest that this molecule contains both right and left-handed conformations under condition of high salt, and thus a B-Z junction.     

Activation of DNA cleavage by dynemicin A in a B-Z conformational junction.

We report here that the DNA strand scission by dynemicin A is not only sequence-specific but also conformation-specific. The salt-induced B----Z conformational transition dramatically enhanced the cleavage by dynemicin A in a B-Z junction region. By contrast, the bleomycin-Fe(II) complex, the elsamicin A-Fe(II) complex, and esperamicin A1 did not induce any preferential DNA cutting in such a DNA structure. The characteristic hyperreactivity of dynemicin A is observed in (dC-dG)8- and (dC-dG)12-inserted DNAs, but not in (dC-dG)5-inserted DNA. These results suggest value in the use of dynemicin A as proof of the existence of a B-Z junction in vivo and also may aid in understanding the structure of B-Z junctions.     

Energetics of B-Z junction formation in a sixteen base-pair duplex DNA

We report analysis of the NaCl-induced B-Z transition in a 16 base-pair duplex DNA with sequence designed such that when NaCl is increased the left half of the molecule undergoes the B-Z transition while the right half remains in the B-form. An equilibrium thermodynamic model based on the body of available published experimental data and the recent theoretical work of Soumpasis, which indicate, in the salt range above approximately 0.9 M-NaCl, the transition free-energy of B-Z conversion in DNA is a linear function of the NaCl concentration, is employed. Analysis of the B-Z transition of the junction-containing molecule indicates the B-Z junction formed in this 16 base-pair DNA is composed of approximately three base-pairs and has a free energy of formation of approximately 4.7 kcal/mol junction. These values for the junction are in excellent agreement with published estimates of B-Z junction size and energy derived from much longer DNA pieces.     

The interface between an alternating CG motif and a random sequence motif displays altered nuclease activity.

Previously we described the B-Z junctions produced in oligomers containing (5meCG)4 segments in the presence of 5.0 M NaCl or 50 uM Co(NH3)6+3 [Sheardy, R.D. & Winkle, S.A., Biochemistry 28, 720-725 (1989); Winkle, S.A., Aloyo, M.C., Morales, N., Zambrano, T.Y. & Sheardy, R.D., Biochemistry 30, 10601-10606 (1991)]. The circular dichroism spectra of an analogous unmethylated oligomer containing (CG)4, termed BZ-IV, in 5.0 M NaCl and in 50 uM Co(NH3)+3 suggest, however, that this oligomer does not form a B-Z hybrid. BZ-IV possesses Hha I sites (CGCG) in the (CG)4 segment and an Mbo I site (GATC) at the terminus of the (CG)4 segment. BZ-IV is equally digestible in the presence and absence of cobalt hexamine by Hha I, further indicating that the structure of BZ-IV is fully B-like under these conditions. The Mbo I cleavage site at the juncture between the (CG)4 segment and the adjacent random segment displays enhanced cleavage by both Mbo I and its isoschizomer Sau3AI in the presence of cobalt hexamine. In addition, exonuclease III digestion of BZ-IV is inhibited at this juncture. Actinomycin inhibits Mbo I activity in the presence of cobalt hexamine but not in the absence. Together, these results suggest that enzymes recognize the interfaces of (CG)n and adjacent random sequences as altered substrates even in the absence of a B-Z junction formation.     

Sequence effects on the relative thermodynamic stabilities of B-Z junction-forming DNA oligomeric duplexes

Circular dichroism (CD) and ultraviolet absorption techniques were employed in characterizing the sequence-dependent thermodynamic stabilities of B-Z junction-forming DNA duplexes. The Watson strand of the duplexes has the general sequence (5meC-G)4-NXYG-ACTG (where N = A or G and XY represents all permutations of pyrimidine bases). Duplexes were generated by mixing stoichiometric amounts of the complementary strands. Circular dichroism studies indicate that each duplex is fully right-handed at low salt (e.g., 115 mM Na+) but undergoes a salt-induced conformational transition to a structure that possesses both left- and right-handed conformations at high salt (4.5 M Na+), and hence a B-Z junction. Optical melting studies of the DNA duplexes at fixed DNA concentration with total Na+ concentration ranging from 15 mM to 5.0 M were determined. A nonlinear dependence of the melting temperature (Tm) on [Na+] was observed. Thermodynamic parameters at Na+ concentrations of 115 mM and 4.5 M with a wide range of DNA concentrations were determined from UV optical melting studies via construction of van't Hoff plots. A change of a single dinucleotide within these duplexes significantly affected the helix stabilities. The experimentally obtained free energies for the duplex to single-strand transitions were in close agreement with predicted values obtained from two different methods.     

Characterization of genomic poly(dT-dG).poly(dC-dA) sequences: structure, organization, and conformation.

Hybridization studies suggest the abundant presence of poly(dT-dG).poly(dC-dA) (TG-element), a potential Z-DNA sequence, in eucaryotic genomes. We have isolated and characterized TG-elements from different locations in the human genome: from randomly isolated clones, associated with the actin gene family, and linked to another repeated element. The results indicate that the following features are typical of these TG-elements: the elements consist of 20 to 60 base pairs of (dT-dG)n.(dC-dA)n, the sequences characterized in our study were not flanked by direct or inverted repeats, the sequences are interspersed rather than in satellite blocks, the elements are not usually associated with other repeated elements, and some of the elements are found near coding sequences or in introns. Studies on the conformation of a genomic TG-element in a supercoiled plasmid indicate several distinct properties of the TG-element: it is in the Z-form only at low ionic strength, S1 nuclease recognizes its Z-form with a marked preference for one of the B-Z junctions, and the sensitive region extends for 20 base pairs near the B-Z junction. In contrast to the result with the supercoiled plasmid, S1 nuclease failed to recognize the TG-element in minichromosomes.     

DNA bending by the bulge defect

Comparative gel electrophoresis measurements were used to characterize DNA bending in molecules containing an extra adenosine on one strand, the so-called bulge defect. We used oligomers containing A6 tracts separated from the bulged base by varying numbers of nucleotides to determine the direction and magnitude of the bulge bend. Helix unwinding by the bulge was determined from the electrophoretic anomaly as a function of the size of the repeated monomers. We conclude that the bulge bend is 21 degrees +/- 3 degrees, primarily in the direction of tilt away from the bulged base. The total helical advance of the DNA at the bulge site is smaller than would be the case if the complementary T were present, corresponding to an unwinding by 25 degrees +/- 6 degrees. These values are in good agreement with the results of NMR and energy minimization studies of the bulged base in double-helical deoxyoligonucleotides [Woodson, S. A., & Crothers, D.M. (1988) Biochemistry 27, 3130-3141]     

Different mechanism for in vitro formation of nucleosome core particles

The interaction of different histone oligomers with nucleosomes has been investigated by using nondenaturing gel electrophoresis. In the presence of 0.2 M NaCl, the addition of the pairs H2A,H2B or H3,H4 or the four core histones to nucleosome core particles produces a decrease in the intensity of the core particle band and the appearance of aggregated material at the top of the gel, indicating that all these histone oligomers are able to associate with nucleosomes. Equivalent results were obtained by using oligonucleosome core particles. Additional electrophoretic results, together with second-dimension analysis of histone composition and fluorescence and solubility studies, indicate that H2A,H2B, H3,H4, and the four core histones can migrate spontaneously from the aggregated nucleosomes containing excess histones to free core DNA. In all cases the estimated yield of histone transfer is very high. Furthermore, the results obtained from electron microscopy, solubility, and supercoiling assays demonstrate the transfer of excess histones from oligonucleosomes to free circular DNA. However, the extent of solubilization obtained in this case is lower than that observed with core DNA as histone acceptor. Our results demonstrate that nucleosome core particles can be formed in 0.2 M NaCl by the following mechanisms: (1) transfer of excess core histones from oligonucleosomes of free DNA, (2) transfer to excess H2A,H2B and H3,H4 associated separately with oligonucleosomes to free DNA, (3) transfer to excess H2A,H2B initially associated with oligonucleosomes to DNA, followed by the reaction of the resulting DNA-(H2A,H2B) complex with oligonucleosomes containing excess H3,H4, and (4) a two-step transfer reaction similar to that indicated in (3), in which excess histones H3,H4 are transferred to DNA before the reaction with oligonucleosomes containing excess H2A,H2B. The possible biological implications of these spontaneous reactions are discussed in the context of the present knowledge of the nucleosome function.     

Determination of the extent of DNA bending by an adenine-thymine tract

We determined the magnitude of the bend induced in DNA by an adenine-thymine tract by measuring the rate of cyclization of DNA oligonucleotides containing phased A tracts. A series of linear multimers with 2-bp single-stranded ends, in which the (A.T)6 tracts are separated by CG2-3C sequences and are positioned 10 and 11 bp apart alternately, were prepared from 21 bp long synthetic duplexed deoxyoligonucleotides. The cyclization rates of the multimers (105-210 bp) and the bimolecular association rate of the 84 bp long multimer were measured in the presence of DNA ligase. From the rate constants of the cyclization and bimolecular association reactions, ring closure probabilities were obtained for the multimers. The systematically bent molecules were simulated by Monte Carlo methods, and the ring closure probabilities were calculated for a given set of junction bend angles. By comparing the calculated values of ring closure probabilities to experimental values and adjusting the junction bend angles to fit experimental values, the extent of bending at the junctions (or the extent of bending for an adenine tract) was determined. We conclude that an A6 tract bends the DNA helix by 17-21 degrees.     

Unpaired bases in the B--Z junction of the supercoiled plasmid

The restriction analysis has been used to establish that O-beta-diethylaminoethylhydroxylamine (OHA) produces modification of unpaired cytidines in the polylinker region adjacent to the Z-insert (dG-dC)10. (dG-dC)10 in the negatively supercoiled plasmid pGC20. The length of the transition region between B- and Z-portions of DNA is not less than 36 bps. The reaction of OHA with the unpaired cytidines in the B-Z junction is a fixing one and produces no secondary despiralling of the neighboring regions. The reaction with DNA proceeds much slower than the one with monomers and single-strand polynucleotides. The structural nonuniformity has been observed, which is manifested in the alternating B and "non-B" form DNA in the B-Z junction. It is suggested that these junctions may contain nucleotide sequences which are stable to violation of the B structure during the change in superhelical density of DNA.     

Methylation of cytosine in the 5-position alters the structural and energetic properties of the supercoil-induced Z-helix and of B-Z junctions

The structural and energetic consequences of cytosine methylation in the 5-position on the supercoil-dependent B-Z equilibrium in alternating dC-dG sequences cloned into recombinant plasmids were investigated. The helical parameters determined with the band shift method for right-handed [10.7 base pairs (bp)/turn] and left-handed (12.8 bp/turn) 5MedC-dG inserts were different from the helical repeat values for unmethylated dC-dG inserts (10.5 bp/turn in the right-handed and 11.5 bp/turn in the left-handed form). We analyzed the thermodynamic parameters delta GBZ (free energy difference per base pair between right-handed and left-handed helix structure), delta Gjx (free energy for formation of one B-Z junction), and b (helix unwinding at a junction region) for varying lengths of dC-dG inserts by two-dimensional gel electrophoresis and application of a statistical mechanics model. A comparison of plasmids fully methylated in vitro with HhaI methylase and their unmethylated counterparts revealed that delta Gjx is not significantly changed by cytosine methylation. However, this base modification results in an approximate 3-fold decrease of delta GBZ and an approximate 2-fold decrease of the unwinding b at B-Z junction regions. Analysis of a pair of related plasmids, each containing two dC-dG blocks, revealed qualitatively different transition behaviors. When the two dC-dG blocks were separated by 95 bp of a mixed sequence, they underwent independent B to Z transitions with separate nucleation events and junction formations. When the two blocks were separated by only a 4 bp GATC sequence, only one nucleation event was necessary, and the Z-helix spread across the nonalternating GATC region.(ABSTRACT TRUNCATED AT 250 WORDS)     

The length of a junction between the B and Z conformations in DNA is three base pairs or less

Recently it has been suggested that double-helical complexes formed between the DNA sequences (CG)n(A)m and their conjugates, (T)m(CG)n, would be candidates for the formation of a B-Z junction in aqueous solution at high salt concentrations [Peticolas et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 2579-2583]. The junction was predicted to occur between a B-type helix in the d(A)m.d(T)m section and a Z-type helix in the self-complementary (CG)n.(CG)n sequence. In this paper we report Raman experiments on the deoxyoligonucleotides d(CGCGCGCGCGCGAAAAA) and d(CGCGCGAAAAA) and their complements. It is found the latter compound cannot be induced into the Z form in saturated salt solution but that the former sequence goes into a B-Z junction at 5.5 M salt. From a comparison of the relative intensity of the Raman conformational marker bands for B and Z DNA for both the A-T and C-G base pairs, it is shown that in 5.5 M NaCl solution none of the A-T base pairs are in the Z form, but nine of the C-G base pairs are in the Z form. The remaining three C-G base pairs are either in the junction or in the B form. Thus, the junction is formed from three or less C-G base pairs. If the solution is made 95 microM with NiCl2, then the entire duplex goes into the Z form and the Raman bands of the adenine are completely changed into those of the Z form.(ABSTRACT TRUNCATED AT 250 WORDS)     

Topological measurement of an A-tract bend angle: effect of magnesium

Sequences of four to six adenine residues, termed A-tracts, have been shown to produce curvature in the DNA double helix. A-tracts have been used extensively as reference standards to quantify bending induced by other sequences as well as by DNA binding proteins when they bind to their sites. However, the ability of an A-tract to serve as such a standard is hampered by the wide variation of values reported for the amount of bend conferred by an A-tract. One experimental condition that differs in these studies is the presence of divalent cation. To evaluate this effect, a new application of a topological method, termed rotational variant analysis, is used here to measure for the first time the effect of the presence of magnesium ion on the bend angle conferred by an A-tract. This method, which has the unique ability to measure a bend angle in the presence or absence of magnesium ion, demonstrates that magnesium ion markedly increases the bend angle. For example, when measured in a commonly used gel electrophoretic buffer, the bend angle conferred by a tract of six adenine residues increases from about 7 degrees in the absence of magnesium ion to 19 degrees in the presence of 3.9 mM magnesium ion. This quantitative demonstration of substantial magnesium ion dependence has several important implications. First, it explains discrepancies among bend values reported in various previous studies, particularly those employing gel electrophoretic versus other solution methods. In addition, these findings necessitate substantial revisions of the conclusions in a large number of studies that have used A-tract DNA as the bend angle reference standard in comparison measurements. Finally, any such future studies employing this comparison methodology will need to use the same sequence analyzed in the original measurements as well as replicate the original measurement conditions (e.g. ionic composition and temperature).     

Effect of P-chirality of internucleotide bonds on B-Z conversion of stereodefined self-complementary phosphorothioate oligonucleotides of the [PS]-d(CG)4 and [PS]-d(GC)4 series

Diastereomerically pure, partially modified (in selected positions) or fully modified phosphorothioate oligomers of the [PS]-d(CG)(4) and [PS]-d(GC)(4) series were investigated with respect to their ability to adopt the left-handed conformation at high sodium chloride concentration. NaCl induces the B-Z transition of [All-S(P)R(P)-PS]-d(CG)(4) with a midpoint of transition at ca. 2 M, which is approximately 1 M less than for unmodified d(CG)(4). Also, [All-R(P)S(P)-PS]-d(GC)(4) at 5 M NaCl converts to the Z form to the extent of ca. 55%, while the unmodified d(GC)(4) counterpart does not convert at all. This enhanced ability of stereodefined phosphorothioate oligomers to adopt the Z conformation is discussed in terms of already known structural factors (hydrogen bonding and water bridges) facilitating the B-Z transition, identified for unmodified d(CG)(n) oligonucleotides. By CD spectroscopy, the [All-S(P)-PS]-d(CG)(4) oligomer at a NaCl concentration higher than 0.01 M adopts a unique conformation as assessed from the presence of an additional negative band centered at 282 nm.     

The Z-Z junction: the boundary between two out-of-phase Z-DNA regions

The boundary between two segments of Z-DNA that differ in the phase of their syn-anti alternation about the glycosidic bond is termed a Z-Z junction. Using chemical probes and two-dimensional gel electrophoresis, we examined a Z-Z junction consisting of the sequence d[(CG)8C(CG)8] inserted into a plasmid and used energy minimization techniques to devise a three-dimensional model that is consistent with the available data. We show that both alternating CG segments undergo the B-Z transition together to form a Z-Z junction. The junction is very compact, displaying a distinctive reactivity signature at the two base pairs at the junction. In particular, the 5' cytosine of the CC dinucleotide at the junction is hyperreactive toward hydroxylamine, and the two guanines of the GG dinucleotide on the complementary strand are less reactive toward diethyl pyrocarbonate than are the surrounding Z-DNA guanines. Statistical mechanical treatment of the 2-D gel data yields a delta G for forming the Z-Z junction equal to 3.5 kcal, significantly less than the cost of a B-Z junction and approximately equal to the cost of a base out of alternation (i.e., a Z-DNA pyrimidine in the syn conformation). The computer-generated model shows little distortion of the Z helix outside of the central two base pairs, and the energy of the structure and the steric accessibility of the reactive groups are consistent with the data.     

Site-specific chemical modification of B-Z junctions in supercoiled DNA as detected by nuclease S1 digestion, inhibition of restriction cleavage and nucleotide sequencing

Structural distortions on the boundary between right-handed and left-handed segments in the superhelical plasmid pPK2 (a derivative of pUC19 containing (dC-dG)n segments cloned into polylinker) were studied by means of chemical probes. Strong osmium tetroxide, pyridine (Os,py) modification of DNA at native superhelical density (sigma) was found in four thymines surrounding the (dC-dG)13 segment. These results correlated with restriction cleavage inhibition (due to modification): BamHI cleavage was strongly inhibited, unlike the neighbouring XbaI and SalI (weak or no inhibition). In the (dC-dG)8 segment considerably weaker modification of the B-Z junctions was observed, accompanied by weak inhibition of BamHI cleavage, while the neighbouring SmaI and KpnI were not affected. Os,py modification of DNA at native sigma was not detected by nuclease S1 cleavage at and (dC-dG)n segment. However, this enzyme recognized and cleaved at the B-Z junction, osmium modified at more negative sigma. The results obtained with the glyoxal and diethyl pyrocarbonate modification support the idea of very narrow B-Z junctions at native sigma.