Left-handed DNA: from Synthetic Polymers to Chromosomes

Tm, Jovin; Lp, McIntosh; Dj, Arndt-Jovin; Da, Zarling; Robert‐Nicoud, Michel; Jh, van de Sande; Kf, Jorgenson; Eckstein, Fritz

doi:10.1080/07391102.1983.10507425

Cited by 181 publications

(123 citation statements)

References 94 publications

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“…In many instances, the loop sizes were quite similar, representing -10% of the length of the genome (see below). Reversal of anti-Z IgG binding by ethidium bromide intercalation or restriction endonuclease cleavage Ethidium bromide induces a reverse Z -B transition in linear and supercoiled DNA molecules (Pohl et al, 1972;DiCapua et al, 1983;Jovin et al, 1983;van de Sande and Jovin, 1981). In closed circular DNA the drug concentration required for effecting Z-B transitions depends on the negative superhelix density DiCapua et al, 1983).…”

Section: Resultsmentioning

confidence: 99%

Electron microscopy of SV40 DNA cross-linked by anti-Z DNA IgG.

Hagen¹,

Zarling²,

Jovin

1985

The EMBO Journal

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Electron microscopy has revealed the specific binding of bivalent anti-Z DNA immunoglobulin G (IgG) to different sites on supercoiled Form I SV40 DNA. The anti-Z IgG links together left-handed regions located within individual or on multiple SV40 DNA molecules at the superhelix density obtained upon extraction. Velocity sedimentation, electrophoresis, and electron microscopy all show that two or more Z DNA sites in the SV40 genome can be intermolecularly cross-linked with bivalent IgG into high mol. wt. complexes. The formation and stability of the intermolecular antibody-DNA complexes are dependent on DNA superhelix density, as judged by three criteria: (1) relaxed circular (Form II) DNA does not react; (2) release of torsional stress by intercalation of 0.25 itM ethidium bromide removes the antibody; and (3) linearization with specific restriction endonucleases reverses antibody binding and DNA cross-linking. Nonimmune IgG does not bind to negatively supercoiled SV40 Form I DNA, nor are complexes observed in the presence of competitive synthetic polynucleotides constitutively in the left-handed Z conformation; B DNA has no effect. Using various restriction endonucleases, three major sites of anti-Z IgG binding have been mapped by electron microscopy to the 300-bp region containing nucleotide sequences controlling SV40 gene expression. A limited number of minor sites may also exist (at the extracted superhelix density). Key words: left-handed Z DNA/enhancer/alternating purine-pyrimidine Introduction We have previously developed an electrophoretic assay which detected left-handed Z DNA sites in SV40 DNA (and other circular genomes) using anti-Z DNA IgG binding to negatively supercoiled Form I molecules (Zarling et al., 1984a(Zarling et al., , 1984b. The formation of DNA-IgG complexes depended on external conditions (ionic strength and temperature), nucleotide sequence, the physical state of the DNA (torsional stress) and the anti-DNA immunoglobulin (specificity, valency and concentration). Changes in electrophoretic migration were attributed to the formation of various oligomeric species; the existence of trimers suggested the expression of at least two Z DNA sites per SV40 DNA.In the present study, SV40 Form I DNA-IgG complexes-were preparatively purified by velocity sedimentation using conditions which optimally stabilize natural Z DNA sequences in protein-

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Section: Resultsmentioning

confidence: 99%

Electron microscopy of SV40 DNA cross-linked by anti-Z DNA IgG.

Hagen¹,

Zarling²,

Jovin

1985

The EMBO Journal

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“…The discovery of the left-handed Z conformation of DNA [l, 21 has resulted in extensive research efforts aimed at understanding its structural details and possible biological significance [3,41. One area of potential biological importance concerns the ability of left-handed DNA to bind and interact with drugs.…”

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confidence: 99%

Chromomycin A₃ binds to left‐handed poly(dG‐m⁵dC)

Shafer¹,

Roques²,

LePecq³

et al. 1988

European Journal of Biochemistry

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The interaction of chromomycin A3 (an antitumor antibiotic) with right-handed and left-handed polynucleotides has been studied by absorbance, fluorescence, circular dichroism, 31P-NMR and 'H-NMR techniques. Binding to either the B form of poly(dG-dC) or the Z form of poly(dG-m5dC) shifts the absorbance maximum to higher wavelength and enhances the fluorescence of the drug. Circular dichroic spectra of solutions containing various concentrations of chromomycin A3 and fixed concentrations of either B or Z polynucleotides show well defined isoelliptic points at similar wavelengths. At the isoelliptic point, the drug complex with B DNA exhibits positive ellipticity while with Z DNA it exhibits negative ellipticity. 31P-NMR spectra of the chromomycin A3 complex with the Z form of poly(dG-m5dC) demonstrate that the Z conformation is retained in the drug complex up to one molecule drug/four base pairs. At Mg2+ concentrations lower than that necessary to stabilize the left-handed conformation of poly(dG-m'dC) alone, 31P analysis shows that chromomycin A3 can bind simultaneously to both the B and Z conformations of poly(dG-m5dC), with no effect on the B -Z equilibrium. These data demonstrate that chromomycin A3 binds to left-handed poly(dG-mSdC) with retention of the lefthanded conformation up to saturating drug concentrations.The discovery of the left-handed Z conformation of DNA [l, 21 has resulted in extensive research efforts aimed at understanding its structural details and possible biological significance [3, 41. One area of potential biological importance concerns the ability of left-handed DNA to bind and interact with drugs. Most studies of non-covalent drug interactions with left-handed DNA reported to date have described drugs which, upon sufficient levels of binding, result in a conversion of the DNA conformation from left-handed to right-handed. The driving force for this Z to B transition lies in the greater affinity of the drug for the right-handed DNA conformation than for the left-handed conformation.Examples of this behavior include ethidium [5 -71, actinomycin D [S, 91, daunomycin [lo], and netropsin [Ill. In addition, several DNA bis-intercalators have been shown to bind to left-handed polynucleotides with substantial affinity and to induce the Z to B transition only when the mode of binding switches from bis-intercalation to mono-intercalation [6, 121.In their study on the properties of the left-handed Z * form of poly(dG-dC) stabilized in the presence of ethanol and MgC12, van de Sande and Jovin [8] reported that mithramycin, an aureolic acid antitumor antibiotic, could bind to it with an affinity only slightly smaller than that for the right-handed conformation. The moderate preference of mithramycin for the right-handed from did lead to a reversal of the Z * conformation, although at almost twice the concentration of ethidium necessary to produce the same change in conformation.Correspondence to R. H. Shafer, Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, Sa...

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“…Nuclease cutting, topisomerase relaxation, or treatment with certain intercalating drugs can induce a Z --B transition in plasmid or cellular DNA and eliminate the binding of specific anti-Z-DNA IgG probes. Small amounts of Z-DNA or potential Z-sequences can be detected by anti-Z-DNA IgG immunofluorescence in nuclear DNA from a variety of fixed cells (1)(2)(3)(4). Though it appears that some alternating purine-pyrimidine sequences can be transcribed in nuclear DNA, it is not known whether transcribed RNA can adopt the left-handed Z-RNA conformation.…”

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confidence: 99%

Cytoplasmic Z-RNA.

Zarling¹,

Calhoun²,

Hardin³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgGs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Preor nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or singlestranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation.Alternating purine-pyrimidine sequences having a strong potential to adopt stable leftghanded Z-conformations (e.g., in negatively supercoiled DNAs) are repetitive elements in prokaryotic and eukaryotic DNA genomes. Some of these sequences are transcribed into RNA. The in vivo conformational states of these nucleic acids and their functions are not currently known (for reviews, see refs. 1 and 2).Z-DNA sequences exist in deproteinized negatively supercoiled plasmids, viral DNAs, and fixed (protein-deplet- A detailed report on the extent of bromine modification used to elicit the antibodies used here and its effect on their immunochemical specificities will be published elsewhere (C.C.H., D.A.Z., S. K. Wolk, W. Ross, and I. Tinoco, Jr., unpublished data). Polynucleotides were extensively dialyzed against TE buffer (10 mM Tris HCl, pH 7.4/0.1 mM EDTA). Polynucleotide kinase labeling was by the procedure of Silberklang et al. (6).Radioimmunoassay (RIA) and Nitrocellulose Filter Binding Assays. Direct RIA or competition assays with unlabeled polymers or drugs were as described (3-5). The standard RIA buffer was 200 mM NaCl/40 mM Tris HCl, pH 7.5/4 mM EDTA. Reaction mixtures (20 ,ul) containing 20 ng of polynucleotide substrate were equilibrated for 5 min at 37°C before adding 5 ,ul of the first antibody (either rabbit or mouse). Primary reaction mixtures were incubated for 1 hr at 37°C; this was followed by addition of 1 ,ul of the appropriate second antibody, either affinity-purified goat anti-rabbit IgG or goat anti-mouse IgG (2.4 mg/ml). After an additional hour at 37°C, immune complexes were washed (three times) with RIA buffer, centrifuged at 15,000 x g (10°C), resuspended, and then assayed for radioactivity in 5 ml of Aquasol-2 (New England Nuclear) in a liquid scintillation counter (Beckman). A machine background of 15-35 cpm was subtracted. Filter-binding assays were performed using Millititer nitrocellulose filters on a vacuum manifold (Millipore). Reactions were as described for RIA, except for the omission of the second antibody, and were terminated by the...

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Left-handed DNA: from Synthetic Polymers to Chromosomes

Cited by 181 publications

References 94 publications

Electron microscopy of SV40 DNA cross-linked by anti-Z DNA IgG.

Electron microscopy of SV40 DNA cross-linked by anti-Z DNA IgG.

Chromomycin A₃ binds to left‐handed poly(dG‐m⁵dC)

Cytoplasmic Z-RNA.

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Left-handed DNA: from Synthetic Polymers to Chromosomes

Cited by 181 publications

References 94 publications

Electron microscopy of SV40 DNA cross-linked by anti-Z DNA IgG.

Electron microscopy of SV40 DNA cross-linked by anti-Z DNA IgG.

Chromomycin A3 binds to left‐handed poly(dG‐m5dC)

Cytoplasmic Z-RNA.

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Resources

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Chromomycin A₃ binds to left‐handed poly(dG‐m⁵dC)