Effects of small neutral osmolytes on the supercoiling free energy and intrinsic twist of p30δ DNA

Abstract

Both theory and experiments are employed to investigate the effects of small neutral osmolytes on the average intrinsic twist (l0), the torsion and bending elastic constants, and the twist energy parameter (ET) that governs the supercoiling free energy. The experimental data for ethylene glycol and acetamide at 37 degrees C suggest, and are interpreted in terms of, a model wherein the DNA exhibits an equilibrium between two distinct conformational states that possess different numbers of bound water molecules and exhibit different intrinsic twists and torsion and bending elastic constants. Expressions are derived to relate the effective ET and l0 to the equilibrium constant, water activity (aw), and number (n) of bound water molecules released per cooperative domain undergoing the two-state transition. The variations of l0 and ET with -ln(aw) are similar for acetamide and ethylene glycol at 37 degrees C. Fitting the theory to those data yields the range n = 103-125 for ethylene glycol and n = 71-113 for acetamide, depending on the assumed value of ET for the dehydrated state. The cooperative domain size of the two-state transition is estimated to exceed about 25-30 base pairs (bp). Between 0 and 19.4 w/v % ethylene glycol, the torsion elastic constant, measured by time-resolved fluorescence polarization anisotropy (FPA), increases by 1.37-fold, whereas the measured ET decreases by 1.15-fold over that same range. The implied decrease in bending rigidity over that range is by a factor of about 0.7. The variations of l0 and ET with increasing -ln(aw) due to added ethylene glycol at 37 degrees C are far smaller than the corresponding variations observed previously at 14 and 15 degrees C. However, at 21 degrees C, upon adding either ethylene glycol or acetamide, l0 and ET initially decline steeply with increasing -ln(aw), with slopes possibly comparable to those seen at 14 and 15 degrees C, but then flatten out and follow curves similar to those at 37 degrees C. Possible origins of such mixed behavior are discussed. The effects of betaine at both 37 and 21 degrees C differ qualitatively and quantitatively in various respects from those of ethylene glycol and acetamide. Upon adding sucrose, l0 initially jumps to higher plateaus at both 37 and 21 degrees C, but its effects on ET cannot be reliably assessed, due to the limited range of -ln(aw).

A Structural Transition in Duplex DNA Induced by Ethylene Glycol

“…These data, which previously extended only to ~20 w/v% osmolyte,17 could be satisfactorily modeled by a two-state transition of independent subunits (or domains) of unknown size. However, the terminal lower plateau of the transition could not be located from those data, and neither the value of (−ln a w ) 1/2 nor the slope, ( ∂E T /∂ (− ln a w )) 1/2 , at the midpoint could be reliably assessed.…”

“…Gel electrophoresis was performed in 0.8 w/v% agarose, as described previously 17. Sufficient (0.08 μg/mL) chloroquine was added to reduce the effective intrinsic twist, and thereby shift the linking differences of all visible topoisomers into the slightly positive range.…”

“…When J=1, equation (18) simplifies to the non-cooperative expression employed previously 17. From equation (18) one obtains the slope,…”

“…Both experiments17, 30–34 and simulations24,35,36 pertaining to long DNAs ( N ≥ 2000 bp) in ~0.1 M monovalent salt indicate that the free energy change to vary the linking difference from Δ m = m − l 0 to Δ l is given by…”

Theory for Spin−Lattice Relaxation of Spin Probes on Weakly Deformable DNA

“…75 For all sequences, the spin probe is incorporated at a position 7 basepairs from the 3′-end, in reference to the spin-labeled strand. The solvent viscosity is calculated from the known sucrose concentration using well-established empirical formulas.…”