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

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 microM ethidium bromide removes the antibody; and (3) linearization with specific restriction endonucleases reverses antibody binding and DNA cross‐linking. Non‐immune 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).