A technique to prepare relatively large quantities (>/=100 microg) of highly positively supercoiled DNA is reported. This uses a recombinant archaeal histone (rHMfB) to introduce toroidal supercoils, and an inexpensive chicken blood extract to relax unrestrained superhelical tension. Preparation of positively supercoiled pUC19 DNA molecules, >50% of which have linking number changes ranging fro...

The mollicute Mycoplasma genitalium causes sexually transmitted disease in humans. It has recently come to widespread public attention through its involvement in pioneering synthetic biology experiments. The 580-kilo-base-pair genome of M. genitalium contains just 470 genes, few of which seem to encode conventional transcription regulators. This raises the important question of how does this si...

The excess linking deficit of plasmid DNA from topoisomerase I-defective bacteria (topA mutants) results mainly from transcription and is commonly ascribed to unbalanced relaxation of transcription-induced twin-supercoiled domains. This defect is aggravated in genes for membrane-binding proteins (such as the tet gene) where anchoring of the transcription complex to the bacterial membrane is tho...

Enhancers are regulatory DNA elements that can activate their genomic targets over a large distance. The mechanism of enhancer action over large distance is unknown. Activation of the glnAp2 promoter by NtrC-dependent enhancer in Escherichia coli was analyzed by using a purified system supporting multiple-round transcription in vitro. The data suggest that DNA supercoiling is an essential requi...

A recent study has analysed the action of bacterial DNA gyrase on a single substrate DNA molecule, discriminating the initial DNA wrapping and subsequent supercoiling steps in the reaction cycle.

Reverse gyrase is a unique hyperthermophile-specific DNA topoisomerase that induces positive supercoiling. It is a modular enzyme composed of a topoisomerase IA and a helicase domain, which cooperate in the ATP-dependent positive supercoiling reaction. Although its physiological function has not been determined, it can be hypothesized that, like the topoisomerase-helicase complexes found in eve...

A unifying theory of the denaturation transition of DNA, driven by temperature T or induced by an external mechanical torque Γ is presented. Our model couples in a geometrical way the hydrogen-bond opening and the untwisting of the helicoidal molecular structure. Using the transfer matrix method, we show that denaturation corresponds to a first-order phase transition from BDNA to dDNA phases an...

Using numerical simulations, we compare properties of knotted DNA molecules that are either torsionally relaxed or supercoiled. We observe that DNA supercoiling tightens knotted portions of DNA molecules and accentuates the difference in curvature between knotted and unknotted regions. The increased curvature of knotted regions is expected to make them preferential substrates of type IIA topois...