Abstract
Cyclization of DNA with sticky ends is commonly used to measure DNA bendability as a function of length and sequence, but how its kinetics depend on the rotational positioning of the sticky ends around the helical axis is less clear. Here, we measured cyclization (looping) and decyclization (unlooping) rates (kloop and kunloop) of DNA with sticky ends over three helical periods (100-130 bp) using single-molecule fluorescence resonance energy transfer (FRET). kloop showed a nontrivial undulation as a function of DNA length whereas kunloop showed a clear oscillation with a period close to the helical turn of DNA (∼10.5 bp). The oscillation of kunloop was almost completely suppressed in the presence of gaps around the sticky ends. We explain these findings by modeling double-helical DNA as a twisted wormlike chain with a finite width, intrinsic curvature, and stacking interaction between the end base pairs. We also discuss technical issues for converting the FRET-based cyclization/decyclization rates to an equilibrium quantity known as the J factor that is widely used to characterize DNA bending mechanics.
Highlights
DNA under physiological conditions constantly undergoes conformational changes due to thermal fluctuations
We show that the looping probability density extracted from this study is in good agreement with the worm-like chain model near 100 bp
The protein complexes that bridge two distal sites of short DNA segments are subjected to a significant amount of bending and torsional stress depending on the loop geometry and size [9, 10]
Summary
DNA under physiological conditions constantly undergoes conformational changes due to thermal fluctuations Among those changes, bending motions coupled with twist can bring distal sites into proximity [1] and impact genome packaging and gene regulation [2, 3]. Some of these processes involve looped DNA segments much shorter than 500 bp, a length regime where the bending energy begins to dominate the free energy of loop formation. The protein complexes that bridge two distal sites of short DNA segments are subjected to a significant amount of bending and torsional stress depending on the loop geometry and size [9, 10]. Measuring looping and unlooping dynamics of short DNA segments can give us insights into the energetics and internal forces that govern loop-associated processes and applications
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