Interactions of anti-poly[d(G-br5C)] IgG with synthetic, viral and cellular Z DNAs

Enzymatically synthesized polyd(G-br5C)] was used to prepare specific polyclonal and monoclonal anti-Z DNA IgGs. The binding specificities of these antibodies were characterized using left-handed polynucleotides with the sequences d(G-x5C)n and d(A-x5C)n.d(G-T)n (mean = aza, methyl, bromo, or iodo). Polyclonal anti-polyd(G-br5C)] IgG binds the convex surface of the Z helix as evidenced by the strong requirement for a methyl or halogen group at the C5 position of cytosine. Little or no anti-polyd(G-br5C)] IgG binding occurs to left-handed DNAs carrying a phosphorothioate substitution in the dGpdC bond or an N-5 aza substitution in the cytosine ring. Anti-polyd(G-br5C)] IgG can stabilize transient Z DNA structures in both polymer families, thereby displacing the equilibrium in solution between the right-and left-handed DNA conformations. Anti-polyd(G-br5C)] IgG binding sites are found in all tested covalently closed circular natural DNAs (Form I) at their extracted negative superhelical densities, but not in any of the corresponding relaxed Form II or linear Form III DNAs. Binding of anti-polyd(G-br5-C)] IgG leads to a reduction in the electrophoretic mobility of Form I DNA (e.g. SV40, phi X174, or pBR322) and to the formation of dimers comprised of the bivalent antibody and two supercoiled Form I DNA molecules. The dimers are converted to monomers by DTT treatment. The formation of IgG-DNA complexes is dependent on external conditions (ionic strength, temperature), the properties of the DNA (torsional stress, sequence), and the immunoglobulin (specificity, valency, and concentration). Higher order oligomeric species, indicative of two or more left-handed segments per DNA molecule are formed in reactions of anti-polyd(G-br5C)] IgG with M13 RF I DNA but not with SV40, pBR322, or phi X174 DNAs. However, oligomers of the latter are generated with other anti-Z DNA IgGs having a broader spectrum of anti-Z DNA reactivity. Conditions which destabilize natural Z sequences in deproteinized supercoiled genomes are: monovalent salt concentrations at or above the 'physiological' range, high temperature, and topological relaxation with DNA gyrase (in the absence of ATP) or with type I topoisomerases. DNA gyrase (plus ATP) catalyses an increase in DNA negative superhelical density which leads to greater anti-Z DNA IgG binding, indicating the formation of additional left-handed regions. Polytene chromosomes of insect larvae bind anti-polyd(G-br5C)] IgG specifically and stably at Z DNA sites. The distribution of this IgG binding differs in certain regions from that displayed by anti-Z DNA IgG probes with other sequence specificities.(ABSTRACT TRUNCATED AT 400 WORDS)