Immunoglobulin recognition of synthetic and natural left-handed Z DNA conformations and sequences

The relative immunogenicities of the polyd(G-C)] and polyd(A-C) · d(G-T)] families of helices have been determined. The specificities of the resultant immunoglobulins have been characterized for recognition of different synthetic and natural left-handed sequences and conformations. Certain modifications of polyd(G-C)] in the sugar-phosphate bacbone and cytosine C-5 potentiate the right(R)-to-left(L) (B → Z) transition under physiological conditions. The resulting polynucleotides, polyd(G^S-C)], polyd(G-io⁵C)], polyd(G-br⁵C)] and polyd(G-m⁵C)], are also highly immunogenic. In contrast, DNAs incapable of assuming the left-handed conformation under physiological salt concentrations are weakly or non-immunogenic. These include unmodified polyd(G-C)] as well as members of the polyd(A-C) · d(G-T)] family of sequences bearing pyrimidine C-5 substitutions (methyl, bromo, iodo). These polynucleotides undergo the R → L isomerization under more stringent ionic and thermal conditions.The specificities of purified polyclonal and monoclonal anti-Z DNA immunoglobulins (IgG) were measured by binding to radiolabeled polynucleotides, by electrophoretic analysis of IgG bound to covalent closed circular DNAs, and by immunofluorescent staining of polytene chromosomes. The salt-induced left-handed forms of polyd(G-C)] and its derivatives (including the cytidine C-5 methyl, bromo, iodo, and N-5 aza substituted polynucleotides) and of the modified polyd(A-C) · d(G-T)] polymers are bound to varying degrees by different antibodies. The patterns of substrate recognition demonstrate the existence of several antigenic domains in left-handed DNAs, including the helix convex surface and the sugar-phosphate backbone. Substitutions in these regions can produce enhancing (required substitutions), neutral, or inhibitory effects on subsequent IgG binding. Additionally, certain modifications of either the convex surface of Z DNA at the C-5 position of cytidine (i.e. a methyl group) or of the backbone (i.e. phosphorothioate substitution) can lead to polymorphic lefthanded conformations that are compatible with antibody binding when present individually but not in combination. The recognition patterns exhibited with DNA substrates from the two DNA families indicate that some, but not all, IgGs show specificity for different nucleotide sequences.The anti-Z DNA IgGs were used to probe for specific left-handed Z DNA determinants on plasmid (e.g. pBR322) or viral (e.g. simian virus 40 (SV40)) DNAs and on the acid-fixed polytene chromosomes of dipteran larvae. At their extracted superhelical density, the negatively supercoiled form I, but not the relaxed, nicked, or linear forms of all tested plasmid and viral DNAs specifically bind sequence-independent anti-Z IgGs. Dimers, trimers and higher oligomers of form I DNA cross-linked by bivalent anti-Z IgGs are formed with numerous (e.g. φX174, SV40, pBR322) genomes. Their occurrence depends upon IgG concentration and specificity, the conditions of ionic strength and temperatures and the DNA genome. The IgG cross-linked DNA multimers are converted to monomers by dithiothreitol reduction. Sequence-independent monovalent anti-Z Fab fragments bind form I DNA but do not generate oligomeric species. Multimers of order >2 indicate the existence of at least two anti-Z Ig binding sites per molecule, as in the case of SV40. IgGs differ in their ability to form stable complexes with some sites on natural DNAs, presumably due to their sequence and conformation binding specificities. A differential binding of these antibodies is also observed in certain bands of polytene chromosomes, such as the telomeric regions that are involved in chromosome associations.