Uses of viral vectors have thus far eclipsed uses of non-viral vectors for gene therapy delivery in the clinic. Viral vectors, however, have certain issues involving genome integration, the inability to be delivered repeatedly, and possible host rejection. Fortunately, development of non-viral DNA vectors has progressed steadily, especially in plasmid vector length reduction, now allowing these tools to fill in specifically where viral or other non-viral vectors may not be the best options. In this review, we examine the improvements made to non-viral DNA gene therapy vectors, highlight opportunities for their further development, address therapeutic needs for which their use is the logical choice, and discuss their future expansion into the clinic

DNA supercoiling is essential for bacterial cell survival. We demonstrated that DNA topoisomerase IV, acting in concert with topoisomerase I and gyrase, makes an important contribution to the steady-state level of supercoiling in Escherichia coli. Following inhibition of gyrase, topoisomerase IV alone relaxed plasmid DNA to a final supercoiling density (ς) of −0.015 at an initial rate of 0.8 links min⁻¹. Topoisomerase I relaxed DNA at a faster rate, 5 links min⁻¹, but only to a ς of −0.05. Inhibition of topoisomerase IV in wild-type cells increased supercoiling to approximately the same level as in a mutant lacking topoisomerase I activity (to ς = −0.08). The role of topoisomerase IV was revealed by two functional assays. Removal of both topoisomerase I and topoisomerase IV caused the DNA to become hyper-negatively supercoiled (ς = −0.09), greatly stimulating transcription from the supercoiling sensitive leu-500promoter and increasing the number of supercoils trapped by λ integrase site-specific recombination.

In a single quantitative study, we measured acrA , acrB , tolC , mdfA , and norE expression in Escherichia coli clinical isolates by using real-time PCR. acrA and acrB overexpression strongly correlated with fluoroquinolone and multidrug resistance; tolC , mdfA , and norE expression did not. The order of abundance of efflux pump transcripts in all fluoroquinolone-susceptible isolates was tolC (highest), then acrA and acrB , and then mdfA and norE. Our findings suggest acrAB overexpression is an indicator of multidrug resistance.

DNA replication and recombination generate intertwined DNA intermediates that must be decatenated for chromosome segregation to occur. We showed recently that topoisomerase IV (topo IV) is the only important decatenase of DNA replication intermediates in bacteria. Earlier results, however, indicated that DNA gyrase has the primary role in unlinking the catenated products of site-specific recombination. To address this discordance, we constructed a set of isogenic strains that enabled us to inhibit selectively with the quinolone norfloxacin topo IV, gyrase, both enzymes, or neither enzyme in vivo. We obtained identical results for the decatenation of the products of two different site-specific recombination enzymes, phage λ integrase and transposon Tn3 resolvase. Norfloxacin blocked decatenation in wild-type strains, but had no effect in strains with drug-resistance mutations in both gyrase and topo IV. When topo IV alone was inhibited, decatenation was almost completely blocked. If gyrase alone were inhibited, most of the catenanes were unlinked. We showed that topo IV is the primary decatenase in vivo and that this function is dependent on the level of DNA supercoiling. We conclude that the role of gyrase in decatenation is to introduce negative supercoils into DNA, which makes better substrates for topo IV. We also discovered that topo IV has an unexpectedly strong DNA relaxation activity that, together with gyrase and topo I, is able to set the supercoiling levels in Escherichia coli.

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.