DNA looping by FokI: the impact of twisting and bending rigidity on protein-induced looping dynamics

Abstract

Protein-induced DNA looping is crucial for many genetic processes such as transcription, gene regulation and DNA replication. Here, we use tethered-particle motion to examine the impact of DNA bending and twisting rigidity on loop capture and release, using the restriction endonuclease FokI as a test system. To cleave DNA efficiently, FokI bridges two copies of an asymmetric sequence, invariably aligning the sites in parallel. On account of the fixed alignment, the topology of the DNA loop is set by the orientation of the sites along the DNA. We show that both the separation of the FokI sites and their orientation, altering, respectively, the twisting and the bending of the DNA needed to juxtapose the sites, have profound effects on the dynamics of the looping interaction. Surprisingly, the presence of a nick within the loop does not affect the observed rigidity of the DNA. In contrast, the introduction of a 4-nt gap fully relaxes all of the torque present in the system but does not necessarily enhance loop stability. FokI therefore employs torque to stabilise its DNA-looping interaction by acting as a ‘torsional’ catch bond.

Repetitive Sequence Variations in the Promoter Region of the Adhesin-Encoding Gene sabA of Helicobacter pylori Affect Transcription

“…The rigidity and bendability of certain regions of DNA have been found to influence proteins' ability to loop DNA (26). The formation of DNA loops is essential in processes such as DNA replication and transcription regulation.…”

“…The formation of DNA loops is essential in processes such as DNA replication and transcription regulation. Thus, changes in sequences that modify the rigidity and bendability of sections of DNA influence the energetics of loop formation associated with these essential processes (26). We hypothesize that indels within Table S1 in the supplemental material) is predicted to be 230 bp based on the annotated sequence of H. pylori strain J99, where the sabA poly(T) tract possesses 18 thymines (31).…”

“…We hypothesized that phase variation in the poly(T) tract results in physical changes in the length of the promoter control region that result in a change in DNA topology and affect the transacting regulatory protein interaction(s) governing sabA expression (25,26).…”

Interplay of Protein Binding Interactions, DNA Mechanics, and Entropy in DNA Looping Kinetics

“…A view of the J-factor within the statistical mechanical framework of looping dynamics suggests that the effects of the polymer chain properties should be confined to influencing the association reaction alone, and most theoretical work on looping dynamics has focused on the unlooped lifetime dependence upon the polymer length and properties (6,8,11,44,45). Dissociation rates have typically been thought of as only involving the separation of the binding surfaces at the interface between them and thus would not involve larger-scale events dependent on polymer conformation (16)(17)(18)(19). With a simple model of a semiflexible polymer and a binding interaction with a finite length scale, we have shown how the J-factor can influence both the looped and unlooped lifetimes.…”

“…This approach captures the effect of the polymer chain on the equilibrium looping probability as the ratio of the rate constants for looping and unlooping. Further experimental work expands the study of DNA looping dynamics to cases where proteins, such as Lac repressor (16)(17)(18) and Fok1 (19), mediate the looping. Several theoretical studies examine the role of the protein on the kinetics (20)(21)(22).…”