In vivo consequences of plasmid topology.

Abstract

The topology and physical chemistry of closed circular DNA molecules are well understood, but the significance for events within living cells is less well appreciated. It has been demonstrated recently1–3 that the torsional constraint which arises from negative supercoiling (that is, reduction of linkage) can induce localized novel secondary structure in isolated plasmid and phage DNA. Inverted repeats adopt hairpin-loop structures not found in relaxed DNA. This structural perturbation might be expected to have functional significance within the living cell, but clearly this requires that the torsional free energy be available for unhindered partition between alterations of twist and writhe. Microheterogeneity in DNA structure has recently attracted considerable interest, especially with regard to left-handed sections of duplex4–6. The inverted repeats identified as sites of hairpin formation are relatively small, with stems of 13 base pairs (bp) or less. Whilst these hairpins could result in a relaxation of ∼10% of the plasmid supercoiling energy, it was of considerable interest to try to construct stem–loop features about 10 times larger so as to study the topological consequences. In the cloning experiment described here, designed to produce direct or inverted 130-bp repeats depending on insertional orientation, no inverse species could be discovered, and deletion events were frequent. It is concluded that the inverted repeat deprives Escherichia coli of its antibiotic resistance. Cruciform adoption by the inverted species can totally relax the torsional constraint in the plasmid. These experiments highlight the importance of topological considerations in the genetics of closed circular DNA, and confirm the availability of torsional constraint in vivo.