Analysis of Z-DNA in fixed polytene chromosomes with monoclonal antibodies that show base sequence-dependent selectivity in reactions with supercoiled plasmids and polynucleotides

Five monoclonal anti-Z-DNA antibodies were characterized with respect to their binding of synthetic nucleic acid polymers and of supercoiled circular plasmid DNA. All of the antibodies reacted only with DNA in the Z-conformation; however, they fell into two classes on the basis of sequence specificity. One class, with broad specificity, reacted well with all sequences in the Z-form, including poly(dG-dC), poly(dG-dm5C), and poly (dG-dBr5C) in linear polymers and poly(dG-dC)n and poly[(dC-dA)n.(dT-dG)n] sequences in supercoiled plasmids. The other class bound only Z-DNA formed by poly(dG-dC). Binding of the monoclonal antibodies specifically to inserts of Z-DNA-forming sequences in plasmids was mapped directly by cross-linking of antibody to the DNA, digestion with restriction nuclease, and electrophoretic analysis of both the unbound fragments and the bound fragments recovered from immune complexes. The monoclonal antibodies were used for indirect immunofluorescence staining of Drosophila polytene chromosomes fixed by two procedures. One procedure yielded chromosomes with Z-specific antibody binding in many interbands, a few specific bands, and parts of some puffs. On chromosomes fixed by the second procedure, antibody staining appeared to follow the DNA concentration, staining all bands brightly. For each fixation procedure, chromosomes showed the same staining pattern with each of the broad specificity monoclonal antibodies that had been seen with polyclonal antibodies. The antibodies that reacted only with poly(dG-dC) and poly (dG-dC)n plasmid inserts did not stain chromosomes fixed by either protocol. We conclude that stretches of poly(dG-dC)n sequences do not contribute significantly to the presence of Z-DNA in fixed polytene chromosomes of Drosophila melanogaster.     

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.     

Isolation of Z-DNA binding proteins from SV40 minichromosomes: evidence for binding to the viral control region

Proteins dissociated from SV40 minichromosomes by increasing NaCl concentration were tested for their binding to Z-DNA [Br-poly(dG-dC)] and B-DNA [poly (dG-dC)]. Z-DNA binding proteins are largely released in 0.2 M NaCl whereas most B-DNA binding proteins are not released until 0.6 M NaCl. Incubation of SV40 minichromosomes with Z-DNA-Sephadex in low salt solution results in proteins with Z-DNA binding activity (PZ proteins). These proteins bind to negatively supercoiled DNAs containing left-handed Z-DNA but not to relaxed DNAs. They compete with anti-Z-DNA antibodies in binding to negatively supercoiled DNAs. The binding is tighter to negatively supercoiled SV40 DNA than to other plasmids, suggesting sequence-specific Z-DNA binding. PZ proteins binding to negatively supercoiled SV40 DNA interfere with cleavage at the Sph I sites, within the 72 bp repeat sequences of the viral control region, but not with cleavage at the Bgl I site, at the origin of replication. Removal of PZ proteins also exposes the Sph I sites in the SV40 minichromosomes while addition of PZ proteins makes the sites inaccessible.     

Specificity of monoclonal anti-Z-DNA antibodies from unimmunized MRL/Mp-lpr/lpr mice

Antibodies reactive with left-handed Z-DNA arise spontaneously in the sera of patients with SLE and rheumatoid arthritis and in autoimmune MRL mice. However, the precise specificity of these autoantibodies has not been established. In this report, we have characterized four monoclonal anti-Z-DNA antibodies from unimmunized MRL/Mp-lpr/lpr mice that do not cross-react with B-DNA and can discriminate between different types of left-handed helices. Two of the monoclonal antibodies (Za and Zi) behaved similarly in that they bound to two forms of Z-DNA (Br-poly(dG-dC).poly(dG-dC) and AAF-poly(dG-dC).poly(dG-dC) but not to two other Z-form DNA (poly(dG-5BrdC).poly(dG-5BrdC) or poly(dG-5MedC).poly(dG-5MedC)). Neither antibody (Za or Zi) bound significantly to B-DNA or to denatured DNA. A third antibody (Ze) exhibited similar binding characteristics for the Z-DNA preparations, but also recognized denatured DNA. In contrast, a fourth antibody (3-7.3) bound preferentially to poly(dG-5BrC).poly(dG-5BrdC) in Z conformation. These results provide the first evidence for anti-Z-DNA autoantibodies in autoimmune mice that do not cross-react with native or denatured DNA and indicate that these antibodies exhibit considerable heterogeneity in their fine binding specificity.     

Immunological detection of B-DNA to Z-DNA transition of polynucleotides by immobilization of the DNA conformation on a solid support

We studied the B-DNA to Z-DNA transition of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) in the presence of NaCl using an enzyme immunoassay. The polynucleotides were coated on microtiter plates at varying concentrations of NaCl and treated with a monoclonal anti-Z-DNA antibody, Z22. The plates were subsequently treated with alkaline phosphatase conjugated polyvalent mouse immunoglobulins and the enzyme substrate, p-nitrophenyl phosphate. The color development due to the enzyme-substrate reaction was quantitated using a microplate autoreader. Our results show that the antibody does not recognize the polynucleotides in the B-DNA conformation and binds strongly to the Z-DNA conformation. A smooth transition curve is obtained at intermediate concentrations of the counterions. From the transition curves, we determined the concentration of the counterions at the midpoint of B-DNA to Z-DNA transition. The midpoint concentrations for poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) are 2.3 and 0.74 M NaCl, respectively. Using the immunological method, we also examined the B-DNA to Z-DNA transition of poly(dG-m5dC).poly(dG-m5dC) in the presence of naturally occurring polyamines. The midpoint concentrations of the polyamines are as follows: putrescine, 2.5 mM; spermidine, 34 microM; spermine, 1.8 microM. The midpoint values determined by the enzyme immunoassay are in good agreement with those determined by circular dichroism and ultraviolet absorption spectroscopic measurements. These results demonstrate that immobilization of a preexisting conformation or a mixture of conformations of DNA on a solid support followed by a titration of the DNA conformations using a monoclonal anti-DNA antibody is an excellent method to study the conformational dynamics of DNA.     

Probing specific molecular conformations with the scanning force microscope. Complexes of plasmid DNA and anti-Z-DNA antibodies.

An anti-Z-DNA IgG antibody was used to probe for the left-handed Z-DNA conformation of a d(CG)11 insert in a negatively supercoiled plasmid DNA (pAN022). The complexes were spread on mica in the presence of a quaternary ammonium detergent benzyldimethylalkylammonium chloride and imaged with a scanning force microscope (SFM). The high affinity anti-Z-DNA antibody was retained even after restriction endonuclease cleavage of the DNA. The two arms in the product molecules had unequal lengths in conformity with the known location of the Z-DNA forming insert. Most complexes exhibited one IgG per DNA molecule. The bound antibodies were up to approximately 35 nm in diameter and extended approximately 2 nm from the mica surface. They were generally in a lateral orientation relative to the DNA, in accordance with prior chemical modification experimental data indicating a bipedal mode of binding for an anti-Z-DNA IgG. However, the SFM images also suggest that the DNA bends to accommodate the two Fab combining regions of the antibody. This study demonstrates the utility of the SFM for investigating conformation-dependent molecular recognition.     

An immunochemical examination of acetylaminofluorene-modified poly(dG-dC) X poly(dG-dC) in the Z-conformation

Immunization of rabbits with a complex of methylated bovine serum albumin and N-2-acetylaminofluorene (AAF)-modified poly(dG-dC) X poly(dG-dC), a polynucleotide that can assume the Z-DNA conformation, yielded several populations of antibodies specific for Z-DNA determinants. The Z-DNA determinants were analyzed by examination of the antisera and of antibody preparations purified on immunoadsorbents. The following was found: AAF-poly(dG-dC) X poly(dG-dC) shared Z-DNA determinants in common with poly(dG-dC) X poly(dG-dC) in 3.0 M NaCl, poly(dG-m5dC) X poly(dG-m5dC) in 1.5 M NaCl, and brominated poly(dG-dC) X poly(dG-dC) in 0.2, 1.5, and 3.0 M NaCl. Included among the antibodies induced by these determinants was a subpopulation whose reaction with brominated poly(dG-dC) X poly(dG-dC) was sensitive to increased ionic strength. Another distinct population of antibodies recognized determinants present on AAF-poly(dG-dC) X poly(dG-dC) but not on the other Z-DNAs. Only a small portion of this population was specific for the AAF moiety; the greater part appeared to recognize Z-DNA-associated conformational characteristics that were unique to AAF-poly(dG-dC) X poly(dG-dC). These findings are consistent with the existence of a continuum of Z-DNA determinants, which might be capable of functioning as recognition signals for regulatory DNA-binding proteins.     

Polyamine-induced B-DNA to Z-DNA conformational transition of a plasmid DNA with (dG-dC)n insert.

We investigated the ability of natural polyamines putrescine, spermidine, and spermine to provoke a left-handed Z-DNA conformation in a recombinant plasmid (pDHg16) with a 23-base pair insert of (dG-dC)n.(dG-dC)n sequences. Using a monoclonal anti-Z-DNA antibody (Z22) and an enzyme-linked immunosorbent assay protocol, we found that spermidine and spermine were capable of converting pDHg16 to the Z-DNA form. The concentrations of spermidine and spermine at the midpoint of the B-DNA to Z-DNA transition were 280 and 5 microM, respectively, in buffer containing 50 mM NaCl, 1 mM sodium cacodylate, and 0.15 mM EDTA, pH 7.4. A plot of ln[Na+] versus ln [spermine4+], where [Na+] is the bulk NaCl concentration and [spermine4+] is the spermine concentration at the midpoint of the B-DNA to Z-DNA transition, gave a straight line with a slope of 1.2. Structural specificity was clearly evident in the efficacy of three spermidine homologs to induce the Z-DNA conformation in pDHg16. Putrescine and acetylspermidines had no effect on the conformation of the plasmid DNA up to a 3 mM concentration. Control experiments with the parental plasmid (pDPL6) showed no binding of the plasmid DNA with Z22. These results indicate that spermidine and spermine are capable of provoking the left-handed Z-DNA conformation in small blocks of (dG-dC)n sequences embedded in a right-handed B-DNA matrix. Since blocks of (dG-dC)n sequences are found in certain native DNAs, conformational alterations of these regions to the Z-DNA form in the presence of polyamines may have important gene regulatory effects.     

Importance of DNA conformation in the reaction with cis-dichlorodiammine platinum (II)

Cis-dichlorodiammine platinum (II) has been reacted with synthetic polynucleotides either in B or in Z conformation. The binding of cis-dichlorodiammine platinum (II) stabilizes the Z conformation when reacted with poly (dG-m⁵dC) ·poly (dG-m⁵dC) in the Z conformation as shown by circular dichroism and by the antibodies to Z-DNA. On the other hand, the binding of cis-dichlorodiammine platinum (II) stabilizes a new conformation when reacted with poly(dG-dC)·poly(dG-dC) or poly (dG-m⁵dC)·poly(dG-m5dC) in the B conformation. The antibodies to Z-DNA bind to these platinated polynucleotides. In rabbits, the injection of platinated poly (dG-dC) poly (dG-dC) induces the synthesis of antibodies which recognize Z-DNA. In low salt conditions, the circular dichroism spectra of these platinated polynucleotides differ from those of B-DNA or Z-DNA. The characteristic³¹P nuclear magnetic resonance spectrum of Z-DNA is not detected. It appears only at high ionic strength, as a component of a more complex spectrum.     

Immunological and spectroscopic studies of poly(dG-dC).poly(dG-dC) modified by cis-diamminedichloroplatinum(II)

The conformational changes induced by the binding of cis-diamminedichloroplatinum(II) to poly(dG-dC).poly(dG-dC) have been studied by reaction with specific antibodies, by circular dichroism and 31P nuclear magnetic resonance. Polyclonal and monoclonal antibodies to Z-DNA bind to platinated poly(dG-dC).poly(dG-dC) at low and high ionic strength. Antibodies elicited in rabbits immunized with the platinated polynucleotide bind to double stranded polynucleotides known to adopt the Z-conformation. At low and high ionic strength the circular dichroism spectrum of platinated poly(dG-dC).poly(dG- dC) does not resemble that of poly(dG-dC).poly(dG-dC) (B or Z conformation). At low ionic strength, the characteristic 31P nuclear magnetic resonance spectrum of the Z-form is not detected. It appears only at high ionic strength, as a component of a more complex spectrum.     

Anti-Z-DNA antibody binding can stabilize Z-DNA in relaxed and linear plasmids under physiological conditions.

It is shown that anti-Z-DNA antibody binding can stabilize sequences of d(CG/GC)n and d(CA/GT)n in the Z-DNA conformation in a plasmid in the complete absence of supercoiling. This effect is quantitated by using antibody preparations of different affinities and varying concentrations. The d(CG/GC)n sequence can be stabilized under physiological conditions. This is the first demonstration that a region of Z-DNA can be stabilized by protein binding in a completely relaxed plasmid under physiological conditions. The antibody-Z-DNA complex in the relaxed plasmid is shown to be an equilibrium state and not a long-lived kinetic intermediate since specific binding of the antibody to linearized plasmids containing Z-forming sequences is observed.     

Formation of Z-DNA in negatively supercoiled plasmids is sensitive to small changes in salt concentration within the physiological range.

Negative supercoiling of the plasmid pBR322 with or without an insert of (dG-dC)n induces the formation of Z-DNA as measured by the binding of antibodies specific for Z-DNA. Increasing the concentration of Na+ (or K+) is shown to inhibit the B to Z-DNA conversion. This may be due to the effect of the cation on the B-Z junction. Using the data for B to Z-DNA conversion of the (dG-dC)n inserts, we have estimated the free energy change per base pair as well as the energy of the B-Z junction. In pBR322, a 14-bp segment [CACGGGTGCGCATG] is believed to form Z-DNA at bacterial negative superhelical densities under salt conditions which are similar to those found in vivo.     

Conformational transitions of polynucleotides in the presence of rhodium complexes

We studied the effects of hexammine and tris(ethylene diamine) complexes of rhodium on the conformation of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) using spectroscopic techniques and an enzyme immunoassay. Circular dichroism spectroscopic measurements showed that Rh(NH3)6(3+) provoked a B-DNA----Z-DNA----psi-DNA conformational transition in poly(dG-dC).poly(dG-dC). Using the enzyme immunoassay technique with a monoclonal anti-Z-DNA antibody, we found that the left-handedness of the polynucleotide was maintained in the psi-DNA form. In addition, we compared the efficacy of Rh(NH3)6(3+) and Rh(en)3(3+) to provoke the Z-DNA conformation in poly(dG-dC).poly(dG-dC) and poly(dG-m5dC.poly(dG-m5dC). The concentrations of Rh(NH3)6(3+) and Rh(en)3(3+) at the midpoint B-DNA----Z-DNA transition of poly(dG-dC).poly(dG-dC) were 48 +/- 2 and 238 +/- 2 microM, respectively. The psi-DNA form of poly(dG-dC).poly(dG-dC) was stabilized at 500 microM Rh(NH3)6(3+). With poly(dG-m5dC).poly(dg-m5dC), both counterions provoked the Z-DNA form at approximately 5 microM and stabilized the polynucleotide in this form up to 1000 microM concentration. These results show that trivalent complexes of Rh have a profound influence on the conformation of poly(dG-dC).poly(dG-dC) and its methylated derivative. Furthermore, the Rh complexes are capable of maintaining the Z-DNA form at concentration ranges far higher than that of other trivalent complexes. Our results also demonstrate that the efficacy of trivalent inorganic complexes to induce the B-DNA to Z-DNA transition of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) is dependent on the nature of the ligand as well as the polynucleotide modification. Differences in charge density and hydration levels of counterions or base sequence- and counterion-dependent specific interactions between DNA and metal complexes might be possible mechanisms for the observed effects.     

Z-DNA-binding proteins from bull testis.

Three Z-DNA-binding proteins of Mr 31, 33 and 58 kD were isolated from mature bull testis. They were obtained in a native state suitable for binding studies. These are the first examples of Z-DNA-binding proteins from a mammalian tissue. Purification involved tissue extraction with 0.35 M NaCl, cation exchange chromatography on CM-Trisacryl M and two consecutive anion exchange FPLC runs on Mono Q. The proteins appeared virtually homogeneous by anion exchange FPLC, SDS polyacrylamide gel electrophoresis and reverse phase HPLC (58 kD protein only). Yields from 50 g of testis tissue were: 31 kD protein, 40 micrograms; 33 kD protein, 100 micrograms; and 58 kD protein, 150 micrograms. Z-DNA binding was determined by Scatchard analysis of filter binding data using brominated poly(dG-dC).poly(dG-dC) as a conformation-specific ligand. Dissociation constants (Kz, in mol nucleotide/liter) were: 31 kD protein, 7 X 10(-7) M; 33 kD protein, 8 X 10(-7) M; 58 kD protein, 6 X 10(-8) M (primary binding site) and 6 X 10(-7) M (secondary binding site). B-DNA binding to poly(dG-dC).poly(dG-dG) was too low for reliable determination under the conditions of assay. This attested to a high degree of conformational specificity of the three proteins. The 58 kD protein bound Z-DNA at the primary site with an affinity almost equivalent to that of a polyclonal anti-Z-DNA antiserum raised in a rabbit (Kz, 4 X 10(-8) M).     

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.     

Direct evidence for the presence of left-handed conformation in a supramolecular assembly of polynucleotides.

Hexammine cobalt(III) chloride (Co(NH3)6(3+) provokes a B-DNA----Z-DNA----psi-DNA conformational transition in poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC). The circular dichroism spectrum of psi-DNA is characterized by a manyfold increase of positive ellipticity in the range of 300-225 nm and the complete absence of a negative peak. In order to ascertain the helical handedness of psi-DNA, we used a recently developed enzyme immunoassay technique. This method consisted of treating the polynucleotides with Co(NH3)6(3+) to convert them to the Z- or psi-DNA forms and immobilizing these conformations on a microtiter plate. The plates were subsequently treated with a monoclonal anti-Z-DNA antibody Z22, alkaline phosphatase conjugated, affinity purified immunoglobulins, and the phosphatase substrate. The enzyme-substrate reaction was monitored by reading the absorbance at 405 nm with a microplate autoreader. The monoclonal anti-Z-DNA antibody had no reactivity to the B-DNA form, but bound strongly to both the Z- and psi-DNA forms, showing that Co(NH3)6(3+)-induced psi-DNA form of the polynucleotides exists in the left-handed Z-DNA conformation.     

Spectrophotometrical and immunochemical studies on the conformational changes in poly(dG-dC).poly(dG-dC) after modification by 4-hydroxyaminoquinoline 1-oxide

Poly(dG-dC).poly(dG-dC) was modified by the reaction with 4-hydroxyaminoquinoline 1-oxide (4HAQO) in the presence of seryl-AMP. The conformations of 4HAQO-modified poly(dG-dC).poly(dG-dC) and of poly(dG-dC).poly(dG-dC) were studied by circular dichroism spectra under various salt concentration conditions. 4HAQO residues to guanine bases are inefficient in inducing the transition of poly(dG-dC).poly(dG-dC) from B-form to Z-form conformation. We have elicited monoclonal antibodies against 4HAQO-poly(dG-dC).poly(dG-dC). They were characterized using enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and binding to supercoiled DNA. These antibodies reacted with 4HAQO-poly(dG-dC).poly(dG-dC) specifically but not with 4HAQO-modified DNA or poly(dG).poly(dC). However, they cross-reacted with N-acetoxy-2-acetylaminofluorene-modified poly(dG-dC).poly(dG-dC) in Z-form conformation. These monoclonal antibodies may recognize a unique conformation in poly(dG-dC).poly(dG-dC) after 4HAQO modification.     

Interactions of H1 and H5 histones with polynucleotides of B- and Z-DNA conformations

Interactions of chicken H1 and H5 histones with poly(dA-dT), poly(dG-dC), and the Z-DNA structure brominated poly(dG-dC) were measured by a nitrocellulose filter binding assay and circular dichroism. At low protein:DNA ratios, both H1 and H5 bound more Z-DNA than B-DNA, and binding of Z-DNA was less sensitive to interference by an increase in ionic strength (to 600 mM NaCl). H5 histone bound a higher percentage of all three polynucleotides than did H1 and caused more profound CD spectral changes as well. For spectral studies, histones and DNA were mixed in 2.0 M NaCl and dialyzed stepwise to low ionic strength. Prepared in this way or by direct mixing in 150 mM NaCl, complexes made with right-handed poly(dG-dC) showed a deeply negative psi spectrum (deeper with H5 than with H1). Complexes of histone and Br-poly(dG-dC) showed a reduction in the characteristic Z-DNA spectral features, with H5 again having a greater effect. Complexes of poly(dA-dT) and H5, prepared by mixing them at a protein:DNA ratio of 0.5, displayed a distinctive spectrum that was not achieved with H1 even at higher protein:DNA ratios. It included a new negative band at 287 nm and a large positive band at 255 nm, giving the appearance of an inverted spectrum relative to spectra of various forms of B-DNA. These findings may reflect an ability of the different lysine-rich histones to cause varying conformational changes in the condensation of chromatin in DNA regions of highly biased base sequence.