Dynamics of Single DNA Looping and Cleavage by Sau3AI and Effect of Tension Applied to the DNA

Abstract

Looping and cleavage of single DNA molecules by the two-site restriction endonuclease Sau3AI were measured with optical tweezers. A DNA template containing many recognition sites was used, permitting loop sizes from approximately 10 to 10,000 basepairs. At high enzyme concentration, cleavage events were detected within 5 s and nearly all molecules were cleaved within 5 min. Activity decreased approximately 10-fold as the DNA tension was increased from 0.03 to 0.7 pN. Substituting Ca(2+) for Mg(2+) blocked cleavage, permitting measurement of stable loops. At low tension, the initial rates of cleavage and looping were similar (approximately 0.025 s(-1) at 0.1 pN), suggesting that looping is rate limiting. Short loops formed more rapidly than long loops. The optimum size decreased from approximately 250 to 45 basepairs and the average number of loops (in 1 min) from 4.2 to 0.75 as tension was increased from 0.03 to 0.7 pN. No looping was detected at 5 pN. These findings are in qualitative agreement with recent theoretical predictions considering only DNA mechanics, but we observed weaker suppression with tension and smaller loop sizes. Our results suggest that the span and elasticity of the protein complex, nesting of loops, and protein-induced DNA bending and wrapping play an important role.

Microscopic Basis for the Mesoscopic Extensibility of Dendrimer-Compacted DNA

“…This would lead to what is usually referred to as DNA looping in the context of protein-DNA interactions. The sawtooth-shaped peaks visible in both the experimental and simulated force extension curves are similar in character to the sawtooth patterns observed experimentally in single-molecule force-extension measurements with known DNA looping proteins (47,48) and serve as potential evidence for such looping in the DNA-dendrimer system. Our simulations uncovered two additional aspects, both of electrostatic nature, which may be of interest for further exploration.…”

Feeling Inter‐ or Intramolecular Interactions with the Polymer Chain as Probe: Recent Progress in SMFS Studies on Macromolecular Interactions

“…[94,95] Their results showed that for all of the two-site enzymes (e.g. A good example of such system is tension-dependent DNA cleavage by restriction endonuclease.…”

“…By using optical tweezers, Smith and co-workers investigated the dependence of cleavage activity on DNA tension for a variety of two-and one-site enzymes. [94,95] Their results showed that for all of the two-site enzymes (e.g. Sau3AI and EcoRII) an external force of 5 pN was sufficient to completely inhibit the reactivity, while the reactivity of one-site enzymes was virtually unaffected.…”

Colloidal particle motion as a diagnostic of DNA conformational transitions

“…A related scheme to TPM allows an intramolecular reaction (such as DNA looping) to take place with the bead free, then applies force to read out the result of the reaction (e.g. loop type [10]); this review will focus only on zero-force TPM experiments.…”