Effect of polyethylene glycol on the supercoiling free energy of DNA

• Published in: Biopolymers 2001-02, Vol.58 (2), p.204-217
• Main Authors: Naimushin, Alexei N., Quach, Ngu, Fujimoto, Bryant S., Schurr, J. Michael
• Format: Article
• Language: English
• Subjects: Circular Dichroism; DNA; DNA, Bacterial - chemistry; DNA, Superhelical - chemistry; effective twist energy parameter; intermolecular condensation; Nucleic Acid Conformation; osmotic exclusion mechanism; Plasmids - chemistry; polyethylene glycol; Polyethylene Glycols; secondary structure; supercoiling free energy; Thermodynamics; topoisomer distribution method; torsion elastic constant
• ISSN: 0006-3525; 1097-0282
• DOI: 10.1002/1097-0282(200102)58:2<204::AID-BIP90>3.0.CO;2-1

The supercoiling free energy of pUC19 DNA [2686 base pairs (bp)] was measured in various concentrations of PEG 8000 (polyethylene glycol; molecular weight 8000) by the topoisomer distribution method. The effective twist energy parameter (ET) that governs the supercoiling free energy declined linearly by 1.9‐fold with increasing w/v % PEG from 0 to 7.5%, which lies below the threshold for intermolecular condensation. In principle, PEG could affect ET either via an osmotic exclusion mechanism or by altering the torsion elastic constant, bending rigidity, or self‐repulsions of the DNA. Possible alterations of the DNA secondary structure and torsion elastic constant were assessed by CD spectroscopy and time‐resolved fluorescence polarization anisotropy of intercalated ethidium. Up to 7.5% PEG, the secondary structure of the DNA remained largely unaltered, as evidenced by (1) the absence of any significant change in the CD spectrum, (2) an extremely small relative decrease (−0.0013) in intrinsic twist, and (3) a negligibly small change in the torsion elastic constant. The observed reduction in ET cannot be ascribed primarily to a decrease in torsion elastic constant, and most likely does not stem from a decrease in bending rigidity either. The decrease in medium dielectric constant due to PEG should increase the self‐repulsions, and thereby increase ET, which is opposite to the observed trend. Instead, the observed decline in ET is attributed to an osmotic exclusion mechanism. The change in molar volume excluded to the PEG (ΔVex), when the linking difference converts from Δl = 0 to Δl = ±1, was determined from the observed ET value and PEG osmotic pressure at each concentration. The experimental ΔVex values agree well with theoretical estimates reckoned for a simple osmotic exclusion model, in which PEG is excluded by hard‐core interactions from a concentric cylindrical volume around every duplex segment. The difference in volume excluded to PEG between the Δl = 0 and the Δl = ±1 topoisomers is attributed entirely to the ∼ 0.7 additional writhe “crossing” of two duplex strands at roughly 90°, which is known to occur in the latter species. When the separation between the duplex centers at the “crossing” was adjusted so that the theoretical estimate of ΔVex matched the experimental value at each PEG concentration, a value near 5.7 nm was obtained in each case. The invariance and plausible magnitude of this mean separation at the crossing provide strong support fo