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

Abstract

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-

A Theoretical Study of Formation of DNA Noncanonical Structures Under Negative Superhelical Stress

“…The probability of cruciformation is especially high in pA03 DNA, which is a fourth part of the ColEl plasmid carrying its main palindrome of 31 bp (22). The formation of Z-form segments in pBR322 DNA and SV40 DNA has been tackled experimentally in a number of studies (23)(24)(25)(26). The data reported in (23-24) for pBR322 are not characterized by a very high level of accuracy and do not contradict the results of our calculations.…”

“…This is ruled out by our calculations: an increased probability of the Z conformation is observed only for 2-3 base pairs adjacement to the a region. Nor do our calculations confirm the conclusion made in (25)(26) to the effect that in SV40 DNA the Z form must first of all appear in the region between residues 1 and 400 which contains the SV40 enhancers ( Figure 2). …”

Cytoplasmic Z-RNA.

“…Furthermore, dimers, trimers, and other multimers were observed. Previously, we have shown that these multimers are formed by the binding of bivalent anti-Z-DNA IgGs to Z-DNA tracts on individual DNA molecules (3,4,7,8). In contrast, there was no reactivity of form I M13 or pBR322 at their extracted negative superhelical densities with C-6, D-4, or normal rabbit IgGs (Fig.…”

A computer aided thermodynamic approach for predicting the formation of Z-DNA in naturally occurring sequences.

“…The question of where Z-DNA is likely to form in naturally occurring DNA sequences has been addressed experimentally by several groups (DiCapua et al, 1983;Nordheim and Rich, 1983;Stockton et al, 1983;Revet et al, 1984; Barton and Raphael, 1985;Hagen et al, 1985;Miller et al, 1983). The approaches used in these studies are conceptually similar; negatively supercoiled closed circular DNA is exposed to antibodies or chemical reagents that show specificity for Z-DNA, and the subsequent binding sites are qualitatively correlated with nearby stretches of purine-pyrimidine alternation.…”