Electronmicroscopy and circular dichroism of the dynamics of the formation and dissolution of supramolecular forms of Z-DNA.

Previous electronmicroscopic studies had shown that N-acetylaminofluorene (AFF)-substituted poly(dG-dC)-poly(dG-dC) in the Z conformation, in 10mM Mg++, condensed into periodically banded, branched structures. We now show that similar structures are seen when poly(dG-dC)-poly(dG-dC) is converted to the Z conformation by heating to 60 degrees C in 1mM Mn++ or to 65 degrees in the presence of 0.5mM Mn++. We demonstrate that these banded structures form in solution, i.e. they are not artifacts of the preparative procedures used for electronmicroscopy, by crosslinking the Z conformers in solution with DL-diepoxybutane (DEB), and then restoring the solution to conditions that favor return to the B conformation. Circular dichroism (CD) and immunochemical studies showed that the Z conformation was maintained and the banded supramolecular structures were still seen by electronmicroscopy. Electronmicroscopy and CID were also used to follow the dissolution of the supramolecular structures by controlled scission of the crosslinks with the eventual return to the short double stranded molecules typical of the B conformers. During this process, supercoiled structures, both toroidal and interwound, were observed. The relationship of the toroids to the banded structure is discussed in the context of two possible structures for the condensed polynucleotide. We conclude that DNA, whether in the B or Z conformation, is extremely flexible in the presence of appropriate counter ions, and we present evidence that earlier estimates of their persistence lengths are too high. The inherent tendency to form condensed, highly organized structures is a property of DNA that could play an important role in its "packaging," and in its functions, and might have been critical for the evolution and replication of early life forms.