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PubMed1990

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.

Animals↗

PubMedDate not supplied

[Comparison of the state of the area between B- and Z-segments of superhelical plasmids in vitro and in situ].

The structure of B-Z junction in a cloned plasmid pGC20 containing a (dG-dC)10 insert at the SmaI site has been studied in vitro and in situ by modifying the DNA with O-beta-diethylaminoethylhydroxylamine (OHA). The latter is an analog of hydroxylamine possessing specificity with respect to unpaired cytidine. Experiments in vitro showed a complicated pattern of inhibiting the restriction hydrolysis of the OHA-modified DNA within the polylinker region of the plasmid. As the duration of the DNA reaction with OHA grows, a gradual increase in the inhibition of restriction is observed at the BamHI site neighboring the Z-insert and at the HindIII site at a distance of about 30 bp from the insert, while an intact segment (containing the SalGI site) is retained in the intermediate region. On passing to the cell level, only the region immediately adjacent to the Z-insert appears to be modified. According to estimates, about 30 to 40% of pGC20 molecules have the (dG-dC)10 insert in the Z-form when modified in situ in 1M OHA, pH 5.0.

Base Sequence↗

PubMed1988

Characterization of anti-Z-RNA polyclonal antibodies: epitope properties and recognition of Z-DNA.

Chemically brominated poly[r(C-G)] [Br-poly[r(C-G)]] containing 32% br8G and 26% br5C was recently shown to contain a 1:1 mixture of A- and Z-form unmodified nucleotides under physiological conditions of temperature, pH, and ionic strength [Hardin, C. C., Zarling, D. A., Puglisi, J. D., Trulson, M. O., Davis, P. W., & Tinoco, I., Jr. (1987) Biochemistry 26, 5191-5199]. Proton NMR results show that more extensive bromination of poly[r(C-G)] (49% br8G, 43% br5C) produces polynucleotides containing greater than 80% unmodified Z-form nucleotides. Using these polynucleotides as antigens, polyclonal antibodies were elicited in rabbits and mice specific for the Z-form of RNA. IgG fractions were purified from rabbit anti-Br-poly[r(C-G)] sera and characterized by immunoprecipitation, nitrocellulose filter binding, and ELISA. Two different anti-Z-RNA IgG specificities were observed. Decreased levels of brominated nucleotides in the immunogen correlated with an increased extent of specific cross-reactivity with Z-DNA. Inoculation of rabbits with polynucleotide immunogens containing 49% br8G and 43% of br5C produced specific anti-Z-RNA IgGs that do not recognize Z-DNA determinants. This suggests that the 2'-OH group is part of the anti-Z-RNA IgG determinant. In contrast, Br-poly[r(C-G)] immunogens containing 32% br8G and 26% br5C produced IgGs that specifically recognize both Z-RNA and Z-DNA. These results show that the bromine atoms are not required for recognition of the Z conformation by the antibodies. The affinity of these anti-Z-RNA IgGs for Z-RNA is about 10-fold higher than for Z-DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

[Detection of left-helical segments in eukaryotic DNA].

The method of DNA binding to nitrocellulose filters was applied to DNA isolated from mouse liver and Ehrlich ascite carcinoma (EAC), calf thymus, and lymphocytes from patients with chronic lymphoid leukemia. In those and phage PM2 DNA the increase in the DNA binding to the filters with a rise in NaCl concentration from 0.5 up to 4.5 M was sigmoidal being suggestive of a conformational transition. No such activity was found in the case of phage lambda or single-stranded DNA. The binding decreased dramatically after mild cleavage of DNA with DNAase I or treatment with phospholipase C or Eco RI and Hin PI restrictases. Incubation of DNA with ethidium bromide led to decrease in the amount of bound DNA. This effect was enhanced with a rise in the dye concentration. The isotherms of ethidium bromide binding to eukaryotic DNA obtained in Scatchard plots by optic titration had a component with a positive slope at low values of r. Bivalent ions (Mg2+, Zn2+) shifting the equilibrium towards the Z-form increased the proportion of macromolecules retained on the filters at NaCl concentrations of 1-3 M. Local changes in the helix conformation were studied with the help of chemical probes: diethylpyrocarbonate (guanine Z-DNA) and osmium-pyridine reagent (pyrimidines of boundary B-Z sites). These probes incorporation into samples of liver DNA, EAC, and lymphocytes resulted in chemical modification of all these samples. Modification of DNA by osmium-pyridine reagent led to inhibition of subsequent restriction by Eco RI restrictase. The data obtained are suggestive of the presence of Z-regions in the B-helix of eukaryotic DNA. A topological model of Z-site stabilization in small superhelical loops of DNA fixed by protein or lipoprotein molecules is proposed.

Animals↗

PubMed1985

Crystal structure of a left-handed RNA tetramer, r(C-br8G)2.

The crystal structure of the modified RNA tetramer, r(C-br8G-C-br8G), was determined by x-ray methods. The crystals are trigonal and belong to the space group P3212. There are three independent tetramers in the unit cell and each forms a left-handed duplex similar to Z-DNA regarding the orientations of the base moiety and the sugar puckerings in guanosine and cytidine. The effect of the additional bromine atom and 2'-hydroxy group on the stabilization of the Z-form structure are also described.

Crystallization↗

PubMed1990

Base dynamics of local Z-DNA conformations as detected by electron paramagnetic resonance with spin-labeled deoxycytidine analogues.

Conformation detection and base dynamics of spin-labeled Z-DNA have been investigated by electron paramagnetic resonance (EPR) spectroscopy. The two synthesized and characterized probes used in this study were C(5)-nitroxide-labeled 2'-deoxycytidine 5'-triphosphates, pppDCAT and pppDCAVAT, which serve as suitable substrates for Micrococcus luteus DNA polymerase. Enzymatic incorporation of these probes into (dG-dC)n yields the EPR-active alternating copolymers (dG-dC,DCAT)n and (dG-dC,-DCAVAT)n. These polymers assume typical B- and Z-DNA conformations under respective low (0.1 M NaCl) and high (4.5 M NaCl) salt conditions, as evidenced by their UV-circular dichroism spectra. The EPR line shape of (dG-dC,DCAT)n in Z-form is unique and significantly different from the B-form EPR spectrum. A similar observation is made for (dG-dC,DCAVAT)n. Thus, the EPR line shapes of these spin-labeled DNAs are indicative of their local conformations. The EPR spectra, analyzed with a previously published motional model [Kao, S.-C., Polnaszek, C.F., Toppin, C.R., & Bobst, A.M. (1983) Biochemistry 22, 5563-5568], indicate tau perpendicular values of 4 and 7 ns for the B- and Z-forms, respectively. Therefore, the base dynamics of Z-DNA are about two times slower than in B-DNA.

Circular Dichroism↗

PubMed1992

A 1H-NMR study of the DNA binding characteristics of thioformyldistamycin, an amide isosteric lexitropsin.

The interaction of thioformyldistamycin, an amide isostere of the naturally occurring antibiotic distamycin A, with a self-complementary decadeoxynucleotide duplex, d(CGCAATTGCG)2, has been examined using a variety of high-field 1H-NMR techniques. The ligand exhibits two forms in solution arising from geometric isomerism due to restricted rotation around the thioformamide bond. Only the thermodynamically more stable Z-form is shown to bind to the oligonucleotide along its minor groove at the central 5'-AATT segment with the end groups of the ligand extending into the flanking GC regions but without any close contact at the amidinium terminus. Cross-peaks involving characteristic intra- and interresidue proton connectivities in the 2D experiments (COSY and NOESY) were employed to assign individual resonances of both strands in the asymmetric DNA-drug complex. The solution structure of the complex was constructed by molecular mechanics calculations based upon initial estimates of drug-DNA NOE contacts and further refined through energy minimization. These results complement previous structural studies on distamycin and other lexitropsins with oligonucleotides. The exchange of the ligand between two equivalent binding sites on the DNA sequence was estimated to occur at 40 s-1 with a free energy of activation of 16.5 kcal.mol-1 at 321-326 K. There was no evidence of formation of a 2:1 drug-oligomer complex, in contrast to the case of the natural product, which is attributed to steric demands of the larger sulfur atom.

Base Sequence↗

PubMed1989

Effects of nucleotide bromination on the stabilities of Z-RNA and Z-DNA: a molecular mechanics/thermodynamic perturbation study.

The structures of ZI- and ZII-form RNA and DNA oligonucleotides were energy minimized in vacuum using the AMBER molecular mechanics force field. Alternating C-G sequences were studied containing either unmodified nucleotides, 8-bromoguanosine in place of all guanosine residues, 5-bromocytidine in place of all cytidine residues, or all modified residues. Some molecules were also energy minimized in the presence of H2O and cations. Free energy perturbation calculations were done in which G8 and C5 hydrogen atoms in one or two residues of Z-form RNAs and DNAs were replaced in a stepwise manner by bromines. Bromination had little effect on the structures of the energy-minimized molecules. Both the minimized molecular energies and the results of the perturbation calculations indicate that bromination of guanosine at C8 will stabilize the Z forms of RNA and DNA relative to the nonbrominated Z form, while bromination of cytidine at C5 stabilizes Z-DNA and destabilizes Z-RNA. These results are in agreement with experimental data. The destabilizing effect of br5C in Z-RNAs is apparently due to an unfavorable interaction between the negatively charged C5 bromine atom and the guanosine hydroxyl group. The vacuum-minimized energies of the ZII-form oligonucleotides are lower than those of the corresponding ZI-form molecules for both RNA and DNA. Previous x-ray diffraction, nmr, and molecular mechanics studies indicate that hydration effects may favor the ZI conformation over the ZII form in DNA. Molecular mechanics calculations show that the ZII-ZI energy differences for the RNAs are greater than three times those obtained for the DNAs. This is due to structurally reinforcing hydrogen-bonding interactions involving the hydroxyl groups in the ZII form, especially between the guanosine hydroxyl hydrogen atom and the 3'-adjacent phosphate oxygen. In addition, the cytidine hydroxyl oxygen forms a hydrogen bond with the 5'-adjacent guanosine amino group in the ZII-form molecule. Both of these interactions are less likely in the ZI-form molecule: the former due to the orientation of the GpC phosphate away from the guanosine ribose in the ZI form, and the latter apparently due to competitive hydrogen bonding of the cytidine 2'-hydroxyl hydrogen with the cytosine carbonyl oxygen in the ZI form. The hydrogen-bonding interaction between the cytidine hydroxyl oxygen and the 5'-adjacent guanosine amino group in Z-RNA twists the amino group out of the plane of the base. This may be responsible for differences in the CD and Raman spectra of Z-RNA and Z-DNA.

Bromine↗

PubMed1990

The crystal structure of d(GTACGTAC) at 2.25 A resolution: are the A-DNA's always unwound approximately 10 degrees at the C-G steps?

The structure of the self-complementary octamer d(GTACGTAC) has been analyzed by a single crystal X-ray diffraction method at 2.25 A resolution. The crystallographic R factor was 0.184 for all 1233 reflections at this resolution. In spite of the alternating purine-pyrimidine sequence, d(GTACGTAC) adopts the A-form conformation rather than the left-handed Z-form. The average helix twist and the mean rise per base pair are 32.1 degrees and 3.18 A, respectively. The d(GTACGTAC) helix is characterized by a wide open major groove and small base-pair tilt (9.7 degrees). The partial unwinding of the helix is observed only at the central pyrimidine-purine C-G step, but not at the other pyrimidine-purine T-A steps. Based on this study and six other X-ray studies, we propose a hypothesis that the A-DNA's are always unwound approximately 10 degrees at the C-G steps. Significant differences in base-pair stacking modes are seen between the purine-pyrimidine step and the pyrimidine-purine step. All deoxyribose rings adopt the C3'-endo conformation. All backbone torsion angles fall into the range expected for the A-DNA form, except for the nucleotide G5, whose alpha and gamma torsion angles adopt the trans, trans conformation instead of the common gauche-, gauche+ conformation.

Base Sequence↗

PubMed1991

DNA hairpin loops in solution. Correlation between primary structure, thermostability and reactivity with single-strand-specific nuclease from mung bean.

Hairpin structures formed by seven DNA inverted repeats have been studied by PAGE, UV(CD)-spectroscopy and nuclease cleavage. The hairpins consisted of (CG)3 stems and loops of 2, 3 and 4 residues. Thermal stabilities (Tm) have been determined in low and high ionic strength buffers, where the hairpins were structured in the B- and Z-DNA form respectively. The thermodynamic parameters of hairpin formation have been obtained by a two-state analysis of the hairpin-coil transitions. It is found that, on increasing the number of bases in the loop from 2 to 3 and 4, the Tms of the B-hairpins decrease, whereas the Tms of the same hairpins in the Z-form increase. This confirms previous evidence (1,2) that in a hairpin molecule the size and structure of the loop are modulated by the conformation of the helical stem. Moreover, B-hairpins with loops comprising 2, 3 and 4 bases have been digested with the single-strand-specific nuclease from mung bean. In our experimental conditions (0 degrees C) the nuclease preferentially cleaves the unbonded nucleotides of the loops. However, the rates of loop hydrolysis, which roughly follow a first-order kinetics, markedly depend on the size of the loop. At a ratio of 3 enzyme units/micrograms DNA, the half-lives of hairpins which are expected to form loops of 4, 3 and 2 residues are 90, 145 and 440 minutes respectively. Thermostability and enzymatic digestion data suggest that two-membered loops can be formed in B-hairpins but not in Z-hairpins.

Base Sequence↗

PubMed1991

Destabilization of the duplex and the high-salt Z-form of poly(dG-methyl5dC) by substitution of ethyl for the 5-methyl group.

The B-to-Z conformational transition of poly(dG-dC) is highly promoted by 5-methyl substitution of the dC moiety, i.e. in poly(dG-methyl5dC). By the synthesis of a new poly(dG-dC) analogue, poly(dG-ethyl5dC), the effect of a longer alkyl-chain substituent of dC on structure and conformation has been studied with ultraviolet absorption melting profiles and circular dichroism spectroscopy. The 5-ethyl substituent in poly(dG-ethyl5dC) destabilizes the duplex structure against thermal denaturation compared with both poly(dG-methyl5dC) and poly(dG-dC). C.d. studies also reveal that for the high-salt B-Z transition of poly(dG-ethyl5dC) a higher NaCl concentration is required than for that of poly(dG-methyl5dC), although much lower than for poly(dG-dC). However low-salt Z-DNA in poly(dG-ethyl5dC) shows unique features, e.g. it needs no divalent cations to be stable. The low-salt B-Z transition of poly(dG-ethyl5dC) can also be observed by the absorption-temperature melting profile, in contrast to both poly(dG-methyl5dC) and poly(dG-dC). The effects of MgCl2 concentration, temperature, acid pH and trifluorethanol on the conformation of poly(dG-ethyl5dC) have also been determined.

Circular Dichroism↗

PubMed1992

9-aminoacridine inhibits the B-Z transition of poly(dA-dT).

9-Aminoacridine is the parent compound of a family of pharmacologically active model substances that bind to DNA through intercalation between base pairs. In the present study we show that 9-aminoacridine inhibits the B-to-Z isomerization of poly(dA-dT) in conditions that otherwise cause it to occur (5 M NaCl and 123 mM Ni(ClO4)2). Higher concentrations of Ni(ClO4)2 (155 mM) are able to induce the Z-form due to the disruption of the drug-polynucleotide interaction by the metal ion. Additionally, the dye reverses the Z-form in certain conditions. Thus, the data from this study indicate that 9-aminoacridine binds preferentially to the B-form of poly(dA-dT).

Aminacrine↗

PubMed1992

Vibrational circular dichroism studies of the A-to-B conformational transition in DNA.

The vibrational circular dichroism (VCD) spectra of several natural DNAs as well as tRNA, poly(dG-dC).poly(dG-dC), and poly(dA-dT).poly(dA-dT) are reported for the base deformation modes in the IR region from 1700 to 1550 cm-1 for the polymers in D2O as well as in high alcohol dehydrating conditions. Spectra of both the B- and A-forms were identified. The A-form DNA VCD, not previously reported, has characteristics that can be found in the VCD spectra of RNAs as would be expected from the similarity of their structures. The VCD is sequence-dependent. Under the dehydrating conditions studied, poly(dA-dT)poly(dA-dT),poly(dA).poly(dT), and a high-A-T fraction natural DNA had a different bandshape from the other DNAs, which was similar to that of poly(rA).poly(rU). Poly(dG-dC).poly-(dG-dC) did not form an A-form in high-alcohol conditions but instead had a VCD spectrum much like that of its high-salt-induced Z-form. Qualitative differences seen experimentally between A- and B-form DNA VCD were suggested by the differences in the coupled oscillator VCD calculated for the two forms.

Animals↗

PubMed1985

Interaction of drugs with Z-DNA: cooperative binding of actinomycin D or actinomine to the left-handed forms of poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) reverses the conformation of the helix.

The interaction of actinomycin D and actinomine with poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC) under B- and Z-form conditions has been investigated by optical and phase partition techniques. Circular dichroism data show that the conformation at the binding site is right-handed, even though adjacent regions of the polymer have a left-handed conformation. Actinomycin D binds in a cooperative manner to poly(dG-dC).poly(dG-dC) under both B-form and Z-form conditions. Analysis of the circular dichroism data shows that 5 +/- 1 base pairs of left-handed poly(dG-dC).poly(dG-dC) in 4.4 M NaCl switch to a right-handed conformation for each bound actinomycin D. When the left-handed form of poly(dG-dC).poly(dG-dC) is stabilized by the presence of 40 microM [Co(NH3)6]Cl3, 25 +/- 5 base pairs switch from a left-handed to a right-handed conformation for each bound actinomycin D. Actinomine binds cooperatively to left-handed poly(dG-dC).poly(dG-dC) in 40 microM [Co(NH3)6]Cl3 and to left-handed poly(dG-m5dC).poly(dG-m5dC) in 2 mM MgCl2. Actinomine does not bind to left-handed poly(dG-dC).poly(dG-dC) in 4.4 M NaCl at concentrations as high as 100 microM. Each bound actinomine converts 11 +/- 3 base pairs of left-handed poly(dG-dC).poly(dG-dC) in 40 microM [Co(NH3)6]Cl3 and 7 +/- 2 base pairs of left-handed poly(dG-m5dC).poly(dG-m5dC) in 2 mM MgCl2. The binding isotherm data also indicate that the binding site has a right-handed conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗

PubMed1987

Stabilization of Z-RNA by chemical bromination and its recognition by anti-Z-DNA antibodies.

Limited chemical bromination of poly[r(C-G)] (32% br8G, 26% br5C) results in partial modification of guanine C8 and cytosine C5, producing a mixture of A- and Z-RNA forms. The Z conformation in the brominated polynucleotide is stabilized at much lower ionic strength than in the unmodified polynucleotide. More extensive bromination of poly[r(C-G)] (greater than 49% br8G, 43% br5C) results in stabilization of a form of RNA having a Z-DNA-like (ZD) CD spectrum in low-salt, pH 7.0-7.5 buffers. Raising the ionic strength to 6 M NaBr or NaClO4 results in a transition in Br-poly[r(C-G)] to a Z-RNA (ZR) conformation as judged by CD spectroscopy. At lower ionic strength Z-DNA-like (ZD) and A-RNA conformations are also present. 1H NMR data demonstrate a 1/1 mixture of A- and Z-RNAs in 110 mM NaBr buffer at 37 degrees C. Nuclear Overhauser effect (NOE) experiments permit complete assignments of GH8, CH6, CH5, GH1', and CH1' resonances in both the A- and Z-forms. GH8----GH1' NOEs demonstrate the presence of both A- and Z-form GH8 resonances in slow exchange on the NMR time scale. The NMR results indicate that unbrominated guanine residues undergo transition to the syn conformation (Z-form). Raman scattering data are consistent with a mixture of A- and Z-RNAs in 110 mM NaCl buffer at 37 degrees C. Comparison with the spectrum of Z-DNA indicates that there may be different glycosidic torsion angles in Z-RNA and Z-DNA [Tinoco, I., Jr., Cruz, P., Davis, P., Hall, K., Hardin, C. C., Mathies, R. A., Puglisi, J. D., Trulson, M. O., Johnson, W. C., & Neilson, T. (1986) in Structure and Dynamics of RNA, pp 55-68, Plenum, New York].(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies↗

PubMed1988

Binding of ethidium ion to left-handed Z-RNA induces a cooperative transition to right-handed RNA at the intercalation site.

The equilibrium binding of the ethidium cation (Etd+) to the right-handed A-form of poly-[r(C-G)], the B-form of poly[d(C-G)], and the left-handed Z-forms of Br-poly[r(C-G)] and Br-poly[d(C-G)] was investigated in 0.22 M NaCl by optical methods. Scatchard analysis indicates that Etd+ intercalates into right-handed forms of poly[r(C-G)] and poly[d(C-G)] in a noncooperative manner. Correlation of Etd+ absorbance binding isotherms and polynucleotide circular dichroism data indicates that drug binding to Br-poly[r(C-G) and Br-poly[d(C-G)] results in cooperative conversion from left-handed Z-forms to right-handed intercalated conformations. Approximate stoichiometries necessary to induce the left- to right-handed transitions are 1 Etd+/9 base pairs (bp) for Z-RNA and 1 Etd+/6 bp for Z-DNA. The apparent limiting binding stoichiometries are approximately 1 Etd+/3 bp for RNA and 1 Etd+/2 bp for DNA. The equilibrium binding constants for binding to the right-handed forms decrease in the order Br-poly[d(C-G)], Br-poly[r(C-G)], poly[d(C-G)], and poly[r(C-G)]. Thermodynamic parameters are obtained by van't Hoff analysis of Etd+ absorbance thermal dissociation data. Enthalpy values for all four polynucleotides are negative and of similar magnitude. Negative entropy values indicate that the binding processes are primarily enthalpically driven.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1991

Potassium permanganate as an in situ probe for B-Z and Z-Z junctions.

The availability of DNA structural probes that can be applied to living cells is essential for the analysis of biological functions of unusual DNA structures adopted in vivo. We have developed a chemical probe assay to detect and quantitate left-handed Z-DNA structures in recombinant plasmids in growing E. coli cells. Potassium permanganate selectively reacts with B-Z or Z-Z junction regions in supercoiled plasmids harbored in the cells. Restriction enzyme recognition sites located at these junctions are not cleaved by the corresponding endonuclease after modification with KMnO4. This inhibition of cleavage allows the determination of the relative amounts of B- and Z-forms of the cloned inserts inside the cell. We have successfully applied this method to monitor the extent of Z-DNA formation in E. coli as a function of the growth phase and mutated topoisomerase or gyrase activities. The assay can in principle be used for any unusual DNA structure that contains a restriction recognition site inside or near the structural alteration. It can be a useful tool to analyze in vivo correlations between DNA structure and gene regulatory events.

DNA Restriction Enzymes↗

PubMed1990

Cytosine methylation enhances Z-DNA formation in vivo.

The influence of cytosine methylation on the supercoil-stabilized B-Z equilibrium in Escherichia coli was analyzed by two independent assays. Both the M.EcoRI inhibition assay and the linking-number assay have been used previously to establish that dC-dG segments of sufficient lengths can exist as left-handed helices in vivo. A series of dC-dG plasmid inserts with Z-form potential, ranging in length from 14 to 74 base pairs, was investigated. Complete methylation of cytosine at all HhaI sites, including the inserts, was obtained by coexpression of the HhaI methyltransferase (M.HhaI) in cells also carrying a dC-dG-containing plasmid. Both assays showed that for all lengths of dC-dG inserts, the relative amounts of B and Z helices were shifted to more Z-DNA in the presence of M.HhaI than in the absence of M.HhaI. These results indicate that cytosine methylation enhances the formation of Z-DNA helices at the superhelix density present in E. coli. The B-Z equilibrium, in combination with site-specific base methylation, may constitute a concerted mechanism for the modulation of DNA topology and DNA-protein interactions.

Chloramphenicol↗