Negative superhelical coiling of covalently closed DNA plasmids facilitates the formation of lefthanded Z-DNA segments. This was demonstrated by binding of antibodies specific for Z-DNA. Plasmid pBR322 and two derivatives from it, pLP32 and pLP014, carrying inserts of alternating CG sequences of 32 bp and 14 bp, respectively, were used. Longer inserts required less negative superhelical density to induce the B-Z transitions. Antibody binding to supercoiled plasmids was also visualized by electron microscopy. Cross-linking of the antibody to the negatively supercoiled plasmid and restriction of the DNA with restriction endonucleases demonstrated that the antibodies combine with the CG insert in pLP32. For pBR322, however, evidence suggests that the antibody combines with a section of DNA containing 14 bases with alternating purine and pyrimidine residues with one residue out of alternation: CACGGGTGCGCATG. These crosslinking studies provide evidence for the binding specificity of the anti-Z-DNA antibodies. On the basis of experimental findings, we have calculated the changes in free energy associated with B-Z transitions in superhelical plasmids.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)