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.     

Yolk proteins from nematodes, chickens, and frogs bind strongly and preferentially to left-handed Z-DNA

Yolk proteins purified from the nematode Caenorhabditis elegans, from the frog Xenopus laevis, and from chicken eggs all have the unexpected property of binding strongly and preferentially to a left-handed Z-DNA probe, brominated poly(dG-dC). We estimate that the nematode proteins bind to Z-DNA with an association constant of at least 10(4) (M-1) and that this association constant is at least 40-50-fold higher than the association constant to B-DNA. Thus, yolk proteins have a higher Z-DNA specificity than most of the Z-DNA binding proteins previously isolated from other sources. Although yolk protein binding to Z-DNA is poorly competed by a wide variety of nucleic acids, the interaction is strongly competed by the phospholipids cardiolipin and phosphatidic acid (500-1000-fold better than by the same mass of B-DNA). We suggest that Z-DNA interacts with the yolk protein phospholipid binding site. In general, our results emphasize the danger of using physical properties to infer biological function. In particular, our results should raise serious questions about the biological relevance of previously isolated Z-DNA binding proteins.     

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.     

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).     

Z-DNA-binding proteins. Identification critically depends on the proper choice of ligands

We have previously isolated from bull testis three proteins of molecular mass 31, 33, and 58 kDa that we have tentatively characterized as high affinity Z-DNA-binding proteins. This inference was based on their preferential binding to brominated poly(dG-dC).poly(dG-dC) in Z-form as opposed to the unbrominated polynucleotide in B-form (Gut, S. H., Bischoff, M., Hobi, R., and Kuenzle, C. C. (1987) Nucleic Acids Res. 15, 9691-9705). By partial amino acid sequencing we have provisionally identified the 31- and 33-kDa proteins as members of the high mobility group 2 and 1 protein families, respectively, whereas the 58-kDa protein has so far remained unidentified (Christen, Th., Bischoff, M., Hobi, R., and Kuenzle, C. C. (1990) FEBS Lett. 267, 139-141). In the present study, we have critically reassessed the binding specificity of these three proteins by using more natural Z- and B-DNA ligands. As such we chose supercoiled and relaxed DNA minicircles containing a d(CG)7 insert in the Z- and B-conformation, respectively. Filter binding tests and gel retardation assays performed with these ligands showed that the three testis proteins either do not discriminate between Z- and B-DNA (31- and 33-kDa proteins) or even have a preference for B-DNA (58-kDa protein). Therefore, we question the validity of using brominated poly(dG-dC).poly(dG-dC) as an indicator of Z-DNA binding.     

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.     

The effect of anti-Z-DNA antibodies on the B-DNA-Z-DNA equilibrium

Four different preparations of rabbit and goat anti-Z-DNA antibodies were examined to determine the effects of antibody binding on the B-DNA-Z-DNA equilibrium. One of the four antibodies, a goat IgG, caused a marked lowering in the ionic strength required for the B-DNA to Z-DNA transition in poly(dG-dC) X poly(dG-dC), shifting the midpoint from 2.25 to 2.0 M NaCl. This IgG had a more prominent high affinity antibody population than did the other goat IgG, which caused little change in the midpoint of this transition. The presence of anti-Z-DNA antibodies also reduced the degree of negative supercoiling required for the formation of Z-DNA in (dG-dC)n sequences inserted into closed circular plasmid DNA. The goat IgG with the more marked effect on the salt-induced transition also had a greater effect in favoring Z-DNA formation in negatively supercoiled plasmids. A shift toward Z-DNA formation was observed in circular dichroism measurements upon antibody binding to poly(dG-dC) X poly(dG-dC) in very low ionic strength solution as well. We propose that the stabilization of Z-DNA by antibody binding in poly(dG-dC) X poly(dG-dC) occurs cooperatively, several antibody molecules binding to a single polymer molecule and stabilizing the entire molecule in Z-DNA through their combined binding energies. The stabilization of Z-DNA by antibody binding in a supercoiled plasmid can be significant, and failure to consider this effect and to choose appropriate conditions for measurement can lead to errors in estimating when Z-DNA will form in response to negative supercoiling.     

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.     

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.     

Preferential binding of the chemical carcinogen N-hydroxy-2-aminofluorene to B-DNA as compared to Z-DNA

The reaction between the chemical carcinogen N-hydroxy-2-aminofluorene and poly (dG-dC) . poly (dG-dC) (B-form), poly (dG-m5dC) . poly (dG-m5dC) (B-or Z-form), poly(dG-br5dC) . poly (dG-br5dC) (Z-form) has been studied. The carcinogen binds covalently to B-DNA but does not bind significantly to Z-DNA. These results are discussed as related to the accessibility, the electrostatic potential and the dynamic structure of DNA. The accessibility and the electrostatic potential of DNA do not explain the difference in reactivity of the carcinogen since a related carcinogen N-acetoxy-N-acetyl-2-aminofluorene binds equally well to both B and Z-DNA. On the other hand, poly (dG-dC) . poly(dG-dC) and poly (dG-br5dC) . poly(dG-br5dC), in presence of ethidium bromide binds equally well to N-hydroxy-2-aminofluorene. It is suggested that the very low binding of this carcinogen to Z-DNA as compared to B-DNA is due to differences in the dynamic structures of these two forms of DNA.     

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.     

Zuotin, a putative Z-DNA binding protein in Saccharomyces cerevisiae.

A putative Z-DNA binding protein, named zuotin, was purified from a yeast nuclear extract by means of a Z-DNA binding assay using [32P]poly(dG-m5dC) and [32P]oligo(dG-Br5dC)22 in the presence of B-DNA competitor. Poly(dG-Br5dC) in the Z-form competed well for the binding of a zuotin containing fraction, but salmon sperm DNA, poly(dG-dC) and poly(dA-dT) were not effective. Negatively supercoiled plasmid pUC19 did not compete, whereas an otherwise identical plasmid pUC19(CG), which contained a (dG-dC)7 segment in the Z-form was an excellent competitor. A Southwestern blot using [32P]poly(dG-m5dC) as a probe in the presence of MgCl2 identified a protein having a molecular weight of 51 kDa. The 51 kDa zuotin was partially sequenced at the N-terminal and the gene, ZUO1, was cloned, sequenced and expressed in Escherichia coli; the expressed zuotin showed similar Z-DNA binding activity, but with lower affinity than zuotin that had been partially purified from yeast. Zuotin was deduced to have a number of potential phosphorylation sites including two CDC28 (homologous to the human and Schizosaccharomyces pombe cdc2) phosphorylation sites. The hexapeptide motif KYHPDK was found in zuotin as well as in several yeast proteins, DnaJ of E.coli, csp29 and csp32 proteins of Drosophila and the small t and large T antigens of the polyoma virus. A 60 amino acid segment of zuotin has similarity to several histone H1 sequences. Disruption of ZUO1 in yeast resulted in a slow growth phenotype.     

Bromination stabilizes poly(dG-dC) in the Z-DNA form under low-salt conditions

Using circular dichroism studies, Pohl & Jovin (1972) [Pohl, F.M., & Jovin, T.M. (1972) J. Mol. Biol. 67, 375-396] demonstrated that poly(dG-dC) undergoes a salt-dependent conformational change characterized by a spectral inversion. The low-salt form corresponds to the right-handed B form of DNA and the high-salt form to the left-handed Z-DNA helix. Modification of poly(dG-dC) by adding bromine atoms to the C8 position of guanine and the C5 position of cytosine residues stabilized this polymer in the Z-DNA form under low-salt conditions. The guanine residues were found to be twice as reactive as the cytosine residues. With a modification of 38% Br8G and 18% Br5C, the polymers formed a stable Z-DNA helix under physiological conditions. The bromination produced spectroscopic features very similar to poly(dG-dC) in 4 M NaCl. However, bromination did not freeze the Z structure as was shown by ethidium bromide intercalation studies. Addition of the dye favored an intercalated B-DNA form. The conversion of B- to Z-DNA leads to profound conformational changes which were also seen by a reduced insensitivity to various exo- and endonucleases. Comparative studies showed that the brominated polymers have a high affinity to nitrocellulose filters. In 1 M NaCl, there was virtually no binding of B-DNA, but a substantial binding of Z-DNA was found even at rather low levels of bromination.(ABSTRACT TRUNCATED AT 250 WORDS)     

Left-handed Z-DNA binding by the recA protein of Escherichia coli

recA binding to left-handed Z-DNA was measured using nitrocellulose filter binding assays with four DNA polymers with defined nucleotide sequences and four recombinant plasmids. Two to 7-fold preferential binding of recA to Z-DNA polymers was observed. Left-handed Z-DNA polymer binding by recA required ATP or its nonhydrolyzable analog, ATP(gamma S), while ADP inhibited binding. Complex formation with both B- and Z-forms was influenced by polymer length; recA bound longer DNAs better. recA binding to recombinant plasmids containing supercoil-stabilized Z-DNA was essentially similar to that found for the control vector; thus, no preferential binding of recA to the Z-form was observed. Comparative experiments with the rec1 protein of Ustilago maydis and the Escherichia coli recA protein were performed. In our hands, recA and rec1 have a similar capacity for binding left-handed Z-DNA polymers and for binding recombinant plasmids containing B- and/or Z-regions. recA contains a left-handed Z-DNA-stimulated ATPase activity. This activity differs from the right-handed B-DNA-stimulated activity since it is less sensitive to increasing pH. The kinetics of ATP hydrolysis in B-DNA/Z-DNA mixing experiments showed that the turnover of the Z-DNA recA complex was slower than for B-DNA suggesting that left-handed Z-DNA is more stably bound by recA. Our results are consistent with the postulate that left-handed Z-DNA is involved in genetic recombination.     

Dual conformational recognition by Z-DNA binding protein is important for the B–Z transition process

Left-handed Z-DNA is radically different from the most common right-handed B-DNA and can be stabilized by interactions with the Zα domain, which is found in a group of proteins, such as human ADAR1 and viral E3L proteins. It is well-known that most Zα domains bind to Z-DNA in a conformation-specific manner and induce rapid B–Z transition in physiological conditions. Although many structural and biochemical studies have identified the detailed interactions between the Zα domain and Z-DNA, little is known about the molecular basis of the B–Z transition process. In this study, we successfully converted the B–Z transition-defective Zα domain, vvZα_E3L, into a B–Z converter by improving B-DNA binding ability, suggesting that B-DNA binding is involved in the B–Z transition. In addition, we engineered the canonical B-DNA binding protein GH5 into a Zα-like protein having both Z-DNA binding and B–Z transition activities by introducing Z-DNA interacting residues. Crystal structures of these mutants of vvZα_E3L and GH5 complexed with Z-DNA confirmed the significance of conserved Z-DNA binding interactions. Altogether, our results provide molecular insight into how Zα domains obtain unusual conformational specificity and induce the B–Z transition.     

The Zβ domain of human DAI binds to Z-DNA via a novel B-Z transition pathway

The human DNA-dependent activator of IFN-regulatory factor (DAI) protein, which activates the innate immune response in response to DNA, contains two tandem Z-DNA binding domains (Zα and Zβ) at the NH(2) terminus. The hZβ(DAI) structure is similar to other Z-DNA binding proteins, although it demonstrates an unusual Z-DNA recognition. We performed NMR experiments on complexes of hZβ(DAI) with DNA duplex, d(CGCGCG)(2), at a variety of protein-to-DNA molar ratios. The results suggest that hZβ(DAI) binds to Z-DNA via an active-di B-Z transition mechanism, where two hZβ(DAI) proteins bind to B-DNA to form the hZβ(DAI)-B-DNA complex; the B-DNA is subsequently converted to left-handed Z-DNA. This novel mechanism of DNA binding and B-Z conversion is distinct from Z-DNA binding of the human ADAR1 protein.     

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.     

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.     

Interaction of the antitumor antibiotic mitomycin C with Z-DNA

Mitomycin C (MC), an antitumor antibiotic, alkylated Z-DNAs such as poly(dG-dC)/Co(NH3)3+(6), poly(dG-m5dC)/Mg2+ and brominated poly(dG-dC) upon reductive activation. Computer-generated energy-minimized molecular models indicated that monofunctional alkylation of Z-DNA at the N2-position of guanine by MC did not distort Z-DNA geometry, but bifunctional alkylation, leading to interstrand crosslinks between two N2-positions of guanine was sterically unfavorable. The above three Z-DNA's were exposed both to monofunctionally and bifunctionally activated MC in separate experiments and the resulting covalent MC-polynucleotide complexes were examined for conformation and for covalent MC-adducts, by circular dichroism (CD) spectroscopy and HPLC analysis of nuclease digests, respectively. Monofunctionally activated MC alkylated all three polynucleotides in their Z-forms, resulting in the same monofunctional N2-guanine adduct as that known to be formed with B-DNA. Upon bifunctional activation of MC, poly(dG-dC/Co(NH3)3+(6) reverted to the B-form and bifunctional (cross-link) adducts were detected, identical again with those formed with B-DNA. Poly(dG-m5dC), however, remained in the Z-form after the alkylation and only a monofunctional adduct could be detected. It was concluded that Z-DNA is subject to monofunctional alkylation by MC but cannot be cross-linked. The latter process occurs only when the Z-DNA is labile enough [as is in the case of poly(dG-dC)] to have some B-form in equilibrium at the site of the first formed monolinked adduct; the cross-linking then occurs at such local B-sites, pulling the overall B in equilibrium Z equilibrium irreversibly to the left. These results are in accord with the predictions from the above modeling. The irreversible "lock" by the MC cross-link on B-DNA may be exploited for probing Z-DNA intermediacy in various DNA functions.     

Solubilization and purification of a membrane-associated 3,3'',5-tri-iodo-L-thyronine-binding protein from rat erythrocytes.

3,3',5-Tri-iodo-L-thyronine (L-T3) binding sites from rat erythrocyte membranes were solubilized in an active form by using the zwitterionic detergent CHAPS or the anionic detergent lauroylsarcosine. The binding protein was successively purified by Sephadex G-200 and affinity chromatography. The purified material retained its binding activity and exhibited high affinity and specificity compared with those displayed in the original membrane. Yield was about 10% of the starting activity. The specific binding activity was enriched by approx. 100-fold, which represents a purity of only 0.1%. Analysis of the purified preparation on SDS/PAGE showed two major protein bands (Mr 64,000 and Mr 50,000), but these could not represent the binding protein since the purity obtained was low. However, affinity-labelling experiments with N-bromoacetyl-L-[125I]T3 in intact membranes showed that two proteins (also with Mr values of 64,000 and 50,000) bound the hormone specifically, suggesting a co-migration of hormone receptors and contaminants on gel electrophoresis.