Jonathan M. Fogg

Structural diversity of supercoiled DNA

By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism. Despite its importance, however, much about supercoiled DNA (positively supercoiled DNA, in particular) remains unknown. Here we use electron cryo-tomography together with biochemical analyses to investigate structures of individual purified DNA minicircle topoisomers with defined degrees of supercoiling. Our results reveal that each topoisomer, negative or positive, adopts a unique and surprisingly wide distribution of three-dimensional conformations. Moreover, we uncover striking differences in how the topoisomers handle torsional stress. As negative supercoiling increases, bases are increasingly exposed. Beyond a sharp supercoiling threshold, we also detect exposed bases in positively supercoiled DNA. Molecular dynamics simulations independently confirm the conformational heterogeneity and provide atomistic insight into the flexibility of supercoiled DNA. Our integrated approach reveals the three-dimensional structures of DNA that are essential for its function.

Bullied no more: When and how DNA shoves proteins around

The predominant protein-centric perspective in protein-DNA-binding studies assumes that the protein drives the interaction. Research focuses on protein structural motifs, electrostatic surfaces and contact potentials, while DNA is often ignored as a passive polymer to be manipulated. Recent studies of DNA topology, the supercoiling, knotting, and linking of the helices, have shown that DNA has the capability to be an active participant in its transactions. DNA topology-induced structural and geometric changes can drive, or at least strongly influence, the interactions between protein and DNA. Deformations of the B-form structure arise from both the considerable elastic energy arising from supercoiling and from the electrostatic energy. Here, we discuss how these energies are harnessed for topology-driven, sequence-specific deformations that can allow DNA to direct its own metabolism.

Transfection of shRNA-encoding Minivector DNA of a few hundred base pairs to regulate gene expression in lymphoma cells

This work illustrates the utility of Minivector DNA, a non-viral, supercoiled gene therapy vector incorporating short hairpin RNA from an H1 promoter. Minivector DNA is superior to both plasmid DNA and small interfering RNA (siRNA) in that it has improved biostability while maintaining high cell transfection efficiency and gene silencing capacity. Minivector DNAs were stable for over 48 h in human serum, as compared with only 0.5 and 2 h for siRNA and plasmid, respectively. Although all three nucleic acids exhibited similar transfection efficiencies in easily transfected adhesion fibroblasts cells, only Minivector DNAs and siRNA were capable of transfecting difficult-to-transfect suspension lymphoma cells. Minivector DNA and siRNA were capable of silencing the gene encoding anaplastic lymphoma kinase, a key pathogenic factor of human anaplastic large cell lymphoma, and this silencing caused inhibition of the lymphoma cells. Based on these results, Minivector DNAs are a promising new gene therapy tool.

Supercoiled Minivector DNA resists shear forces associated with gene therapy delivery.

Supercoiled DNAs varying from 281 to 5302 bp were subjected to shear forces generated by aerosolization or sonication. DNA shearing strongly correlated with length. Typical sized plasmids (⩾3000 bp) degraded rapidly. DNAs 2000–3000 bp persisted ∼10 min. Even in the absence of condensing agents, supercoiled DNA <1200 bp survived nebulization, and increased forces of sonication were necessary to shear it. Circular vectors were considerably more resistant to shearing than linear vectors of the same length. DNA supercoiling afforded additional protection. These results show the potential of shear-resistant Minivector DNAs to overcome one of the major challenges associated with gene therapy delivery.

Differences Between Positively and Negatively Supercoiled DNA that Topoisomerases May Distinguish

In all living cells, DNA is homeostatically underwound relative to its lowest energy conformation, resulting in egative supercoiling. This underwinding of DNA is critical to the metabolism of DNA and, thus, is vital to cell survival. Enzymes called topoisomerases regulate and maintain the supercoiled state of DNA and are critical to the successful replication of the genome. These enzymes are major targets for drugs used in the treatment of bacterial infections and cancer. One puzzling phenomenon of the topoisomerase mechanism is how these enzymes, orders of magnitude smaller than their substrate, can search, recognize and act at a local level to affect global DNA topology. While the homeostatic state of DNA supercoiling in cells is negative, both positive and negative supercoils exist transiently. Because of the right-handed nature of the DNA helix, the positive and negative supercoils are not equivalent. Several computational and theoretical models have been developed in an effort to describe the features of both positively and negatively supercoiled DNA. These models have accurately predicted some of the phenomena observed in vivo. However, the over-simplifying assumptions cannot account for the different biological activities of positively and negatively supercoiled DNA. This review will discuss the models in place and the mathematical and energetic properties of this elegant molecule and the “machines that push it around.”

Influence of DNA sequence on the structure of minicircles under torsional stress

Abstract The sequence dependence of the conformational distribution of DNA under various levels of torsional stress is an important unsolved problem. Combining theory and coarse-grained simulations shows that the DNA sequence and a structural correlation due to topology constraints of a circle are the main factors that dictate the 3D structure of a 336 bp DNA minicircle under torsional stress. We found that DNA minicircle topoisomers can have multiple bend locations under high torsional stress and that the positions of these sharp bends are determined by the sequence, and by a positive mechanical correlation along the sequence. We showed that simulations and theory are able to provide sequence-specific information about individual DNA minicircles observed by cryo-electron tomography (cryo-ET). We provided a sequence-specific cryo-ET tomogram fitting of DNA minicircles, registering the sequence within the geometric features. Our results indicate that the conformational distribution of minicircles under torsional stress can be designed, which has important implications for using minicircle DNA for gene therapy.

Erratum: Structural diversity of supercoiled DNA

Nature Communications, 6: Article number: 8440 (2015); Published: 12 October 2015; Updated: 29 October 2015 The original version of this Article contained an error in the spelling of the author Daniel J. Catanese Jr, which was incorrectly given as Daniel J. Catanese. This has now been corrected in both the PDF and HTML versions of the Article.

TopA, the Sulfolobus solfataricus topoisomerase III, is a decatenase

Abstract DNA topoisomerases are essential enzymes involved in all the DNA processes and among them, type IA topoisomerases emerged as a key actor in the maintenance of genome stability. The hyperthermophilic archaeon, Sulfolobus solfataricus, contains three topoisomerases IA including one classical named TopA. SsoTopA is very efficient at unlinking DNA catenanes, grouping SsoTopA into the topoisomerase III family. SsoTopA is active over a wide range of temperatures and at temperatures of up to 85°C it produces highly unwound DNA. At higher temperatures, SsoTopA unlinks the two DNA strands. Thus depending on the temperature, SsoTopA is able to either prevent or favor DNA melting. While canonical topoisomerases III require a single-stranded DNA region or a nick in one of the circles to decatenate them, we show for the first time that a type I topoisomerase, SsoTopA, is able to efficiently unlink covalently closed catenanes, with no additional partners. By using single molecule experiments we demonstrate that SsoTopA requires the presence of a short single-stranded DNA region to be efficient. The unexpected decatenation property of SsoTopA probably comes from its high ability to capture this unwound region. This points out a possible role of TopA in S. solfataricus as a decatenase in Sulfolobus.