DNA supercoiling: A regulatory signal for the λ repressor

Abstract

Topoisomerases, polymerases, and the chirality introduced by the binding of histones or nucleoid-associated proteins affect DNA supercoiling in vivo. However, supercoiling is not just a by-product of DNA metabolism. Supercoiling is an indicator of cell health, it modifies the accessibility of chromatin, and coordinates the transcription of genes. This suggests that regulatory, proteinmediated loops in DNA may sense supercoiling of the genome in which they are embedded. The λ repressor (CI) maintains the quiescent (lysogenic) transcriptome of bacteriophage λ in infected Escherichia coli. CI-mediated looping prevents overexpression of the repressor protein to preserve sensitivity to conditions that trigger virulence (lysis). Experiments were performed to assess how well the CI-mediated DNA loop traps superhelicity and determine whether supercoiling enhances CI-mediated DNA looping. CI oligomers partitioned plasmids into topological domains and prevented the passage of supercoiling between them. Furthermore, in single DNA molecules stretched and twisted with magnetic tweezers, levels of superhelical density confined in CI-mediated DNA loops ranged from −15% or +11%. Finally, in DNA under tensions that may occur in vivo, supercoiling lowered the free energy of loop formation and was essential for DNA looping. Supercoiling-enhanced looping can influence the maintenance of lysogeny in the λ repressor system; it can encode sensitivity to the energy level of the cell and creates independent topological domains of distinct superhelical density.DNA looping | DNA supercoiling | bacteriophage λ repressor | magnetic tweezer | transcription

DNA supercoiling is a fundamental regulatory principle in the control of bacterial gene expression

“…This dependency connects DNA gyrase activity to the metabolic status of the bacterium. The negative supercoils are removed by DNA topoisomerase I through an ATPindependent mechanism; the ATP-dependent DNA topoisomerase IV can also relax negative supercoils have their freedom to diffuse impeded by diffusion barriers in the chromosome, including those due to other moving polymerases and their associated local supercoiling domains (Leng and McMacken 2002) or by barriers erected by proteins capable of bridging different parts of a DNA molecule (Ding et al 2014;Fulcrand et al 2016). The local supercoiling created by the movement of the polymerases can be eliminated by the action of topoisomerases and this is probably the most common solution employed under physiological conditions, with DNA gyrase relaxing the overwound DNA ahead of the moving polymerase and DNA topoisomerase I relaxing the negatively supercoiled DNA behind (Koster et al 2010) (Fig.…”

Platinum-Based Drugs and DNA Interactions Studied by Single-Molecule and Bulk Measurements

“…Since their invention (28)(29)(30), MT techniques have often been used to analyze the interactions between DNA and DNA ligands, including small drug molecules (25,31,32) and proteins (33)(34)(35)(36)(37)(38). MT can be used to apply controlled force and torsion to single DNA molecules and enables measurement of the influence of such nanomechanical stresses on the DNA structure, primarily through the assessment of end-to-end distance at nanometric resolution.…”

Supercoiling biases the formation of loops involved in gene regulation

“…4; figs. 2 and S5 and table 1 in Ding et al 2014) and resisted very high levels of torque. Quite frequently the linking number of the looped segment corresponded to the linking number change mechanically imposed on the entire DNA tether.…”

“…Time-resolved observation following the nicking of one domain with a large quantity of restriction enzyme revealed a relatively slow rate at which supercoils escaped from the intact domain through the protein-mediated loop closure and into the nicked domain, Mechanically introducing about 17 negative supercoils did not significantly change the near-zero extension, and no further gyrase activity was observed when 27 positive supercoils were introduced where they instantaneously relax by twisting. Thus, CI is an effective barrier to the diffusion of supercoiling (Ding et al 2014). Negative supercoiling of DNA under tension enhanced the probability of loop formation and the stability of the loop.…”

“…Negative supercoiling of DNA under tension enhanced the probability of loop formation and the stability of the loop. Tension opposes juxtaposition of the closure sites, and loops larger than 400 bp do not form spontaneously in torsionally relaxed DNA (Ding et al 2014). Using magnetic tweezers to unwind DNA tethers lowered the free energy of loop formation until it became spontaneous.…”