Contributions of supercoiling to Tn3 resolvase and phage Mu Gin site-specific recombination

Members of the resolvase/invertase family of site-specific recombinases require supercoiled substrates containing two recombination sites. To dissect the roles of supercoiling in recombination by the Tn3 and γδ resolvases and the phage Mu Gin invertase, we used substrates that provided some but not all of the topological features of the standard substrate. We divided the Tn3 resolvase reaction into two stages, synapsis and postsynapsis. We found the contributions of supercoiling to each stage were distinct, since substrate catenation in the absence of supercoiling or low levels of substrate supercoiling were sufficient for synapsis but not postsynapsis. Using structural and functional topological analyses, we verified that the resolvase synaptic complexes with nicked catenanes were recombination intermediates. The requirement for supercoiling was even less stringent for the γδ resolvase, which recombined nicked catenanes about half as well as it did supercoiled substrates. Gin recombination of catenanes occurred even if the recombinational enhancer was on a nicked ring, as long as both crossover sites were on a supercoiled ring. Therefore, supercoiling is required at the Gin crossover sites but not at the enhancer. We conclude that solely conformational effects of supercoiling are required for resolvase synapsis and the function of the Gin enhancer, but that a torsional effect, probably double helix unwinding, is needed for Tn3 resolvase postsynapsis and at the Gin recombination sites

Genomic transcriptional response to loss of chromosomal supercoiling in Escherichia coli

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Background The chromosome of Escherichia coli is maintained in a negatively supercoiled state, and supercoiling levels are affected by growth phase and a variety of environmental stimuli. In turn, supercoiling influences local DNA structure and can affect gene expression. We used microarrays representing nearly the entire genome of Escherichia coli MG1655 to examine the dynamics of chromosome structure. Results We measured the transcriptional response to a loss of supercoiling caused either by genetic impairment of a topoisomerase or addition of specific topoisomerase inhibitors during log-phase growth and identified genes whose changes are statistically significant. Transcription of 7% of the genome (306 genes) was rapidly and reproducibly affected by changes in the level of supercoiling; the expression of 106 genes increased upon chromosome relaxation and the expression of 200 decreased. These changes are most likely to be direct effects, as the kinetics of their induction or repression closely follow the kinetics of DNA relaxation in the cells. Unexpectedly, the genes induced by relaxation have a significantly enriched AT content in both upstream and coding regions. Conclusions The 306 supercoiling-sensitive genes are functionally diverse and widely dispersed throughout the chromosome. We propose that supercoiling acts as a second messenger that transmits information about the environment to many regulatory networks in the cell.Published versio

Characterization of Knots and Links Arising From Site-specific Recombination on Twist Knots

We develop a model characterizing all possible knots and links arising from recombination starting with a twist knot substrate, extending previous work of Buck and Flapan. We show that all knot or link products fall into three well-understood families of knots and links, and prove that given a positive integer $n$, the number of product knots and links with minimal crossing number equal to $n$ grows proportionally to $n^5$. In the (common) case of twist knot substrates whose products have minimal crossing number one more than the substrate, we prove that the types of products are tightly prescribed. Finally, we give two simple examples to illustrate how this model can help determine previously uncharacterized experimental data.Comment: 32 pages, 7 tables, 27 figures, revised: figures re-arranged, and minor corrections. To appear in Journal of Physics

Coiling Instability of Multilamellar Membrane Tubes with Anchored Polymers

We study experimentally a coiling instability of cylindrical multilamellar stacks of phospholipid membranes, induced by polymers with hydrophobic anchors grafted along their hydrophilic backbone. Our system is unique in that coils form in the absence of both twist and adhesion. We interpret our experimental results in terms of a model in which local membrane curvature and polymer concentration are coupled. The model predicts the occurrence of maximally tight coils above a threshold polymer occupancy. A proper comparison between the model and experiment involved imaging of projections from simulated coiled tubes with maximal curvature and complicated torsions.Comment: 11 pages + 7 GIF figures + 10 JPEG figure

Force-Extension Relations for Polymers with Sliding Links

Topological entanglements in polymers are mimicked by sliding rings (slip-links) which enforce pair contacts between monomers. We study the force-extension curve for linear polymers in which slip-links create additional loops of variable size. For a single loop in a phantom chain, we obtain exact expressions for the average end-to-end separation: The linear response to a small force is related to the properties of the unstressed chain, while for a large force the polymer backbone can be treated as a sequence of Pincus--de Gennes blobs, the constraint effecting only a single blob. Generalizing this picture, scaling arguments are used to include self-avoiding effects.Comment: 4 pages, 5 figures; accepted to Phys. Rev. E (Brief Report

Analysis of the eukaryotic topoisomerase II DNA gate: a single-molecule FRET and structural perspective

Type II DNA topoisomerases (topos) are essential and ubiquitous enzymes that perform important intracellular roles in chromosome condensation and segregation, and in regulating DNA supercoiling. Eukaryotic topo II, a type II topoisomerase, is a homodimeric enzyme that solves topological entanglement problems by using the energy from ATP hydrolysis to pass one segment of DNA through another by way of a reversible, enzyme-bridged double-stranded break. This DNA break is linked to the protein by a phosphodiester bond between the active site tyrosine of each subunit and backbone phosphate of DNA. The opening and closing of the DNA gate, a critical step for strand passage during the catalytic cycle, is coupled to this enzymatic cleavage/religation of the backbone. This reversible DNA cleavage reaction is the target of a number of anticancer drugs, which can elicit DNA damage by affecting the cleavage/religation equilibrium. Because of its clinical importance, many studies have sought to determine the manner in which topo II interacts with DNA. Here we highlight recent single-molecule fluorescence resonance energy transfer and crystallographic studies that have provided new insight into the dynamics and structure of the topo II DNA gate