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).
Hagen
F K
FK
Zarling
D A
DA
Jovin
T M
TM
D000906:Antibodies; D002874:Chromosome Mapping; D003432:Cross-Linking Reagents; D004270:DNA, Circular; D004279:DNA, Viral; D004996:Ethidium / Q000494:pharmacology; D008854:Microscopy, Electron; D009690:Nucleic Acid Conformation / Q000187:drug effects; D013539:Simian virus 40 / Q000648:ultrastructure*
D000906:Antibodies; D003432:Cross-Linking Reagents; D004270:DNA, Circular; D004279:DNA, Viral; D004996:Ethidium