Condensation of plasmids enhanced by Z-DNA conformation of d(CG)n inserts.

摘要

DNA molecules collapse into compact structures in the presence of multivalent cations. To probe the possible importance of supercoiling and conformational effects, pUC18 plasmids (2686 bp) were modified by inserting 12-bp and 20-bp alternating d(CG)n sequences, which are capable of converting to a left-handed Z-conformation under appropriate conditions, into the polycloning region. Condensation was induced by rapid addition of hexaammine cobalt(III) [Co(NH3)6(3+)] and monitored by laser light scattering and electron microscopy. Light scattering shows that plasmids with longer d(CG)n inserts condense more extensively at natural superhelical densities. Electron microscopy indicates that the morphological distribution of condensed d(CG)n-containing plasmids changes as a function of Co(NH3)6(3+) concentration. At lower Co(NH3)6(3+) concentration, the proportion of rods is higher, and at higher Co-(NH3)6(3+) concentration, most of the condensates have the form of toroids. In addition, the inner radii of thetoroids are much smaller relative to condensed pUC18 under the same conditions. Enzymatic analysis and chemical probing show that the d(CG)n inserts in naturally supercoiled plasmids have extensively converted from B-form to Z-form in the presence of Co(NH3)6(3+) at the upper range of concentrations under which condensation occurs. To determine whether the enhanced condensation of d(CG)n-containing plasmids results from the change of superhelical density due to the B-Z transition, we treated wild-type pUC18 molecules with topoisomerase I and varying amounts of ethidium bromide to generate a range of supercoil densities. Light scattering indicates that supercoiling did not affect the condensation process.(ABSTRACT TRUNCATED AT 250 WORDS)