Hydrophobic catalysis and a potential biological role of DNA unstacking induced by environment effects

Abstract

Hydrophobic base stacking is a major contributor to DNA double-helix stability. We report the discovery of specific unstacking effects in certain semihydrophobic environments. Water-miscible ethylene glycol ethers are found to modify structure, dynamics, and reactivity of DNA by mechanisms possibly related to a biologically relevant hydrophobic catalysis. Spectroscopic data and optical tweezers experiments show that base-stacking energies are reduced while base-pair hydrogen bonds are strengthened. We propose that a modulated chemical potential of water can promote "longitudinal breathing" and the formation of unstacked holes while base unpairing is suppressed. Flow linear dichroism in 20% diglyme indicates a 20 to 30% decrease in persistence length of DNA, supported by an increased flexibility in single-molecule nanochannel experiments in poly(ethylene glycol). A limited (3 to 6%) hyperchromicity but unaffected circular dichroism is consistent with transient unstacking events while maintaining an overall average B-DNA conformation. Further information about unstacking dynamics is obtained from the binding kinetics of large thread-intercalating ruthenium complexes, indicating that the hydrophobic effect provides a 10 to 100 times increased DNA unstacking frequency and an "open hole" population on the order of 10^-2 compared to 10^-4 in normal aqueous solution. Spontaneous DNA strand exchange catalyzed by poly(ethylene glycol) makes us propose that hydrophobic residues in the L2 loop of recombination enzymes RecA and Rad51 may assist gene recombination via modulation of water activity near the DNA helix by hydrophobic interactions, in the manner described here. We speculate that such hydrophobic interactions may have catalytic roles also in other biological contexts, such as in polymerases.

Keywords: DNA; DNA polymerase; RecA; hydrophobic catalysis; threading intercalation.

Fig. 1.

Flow LD confirms B-DNA and indicates shorter persistence length in diglyme. (A) DNA LD spectra (720 rpm; see SI Appendix, Figs. S14–S16 for all speeds) with wavelength-independent LD^r is consistent with bases near perpendicular to helix axis in diglyme and glycerol. (B) LD^r in diglyme is less negative than in glycerol of the same viscosity. Hence, DNA persistence length is shortened in diglyme. Extrapolation to zero flow gradient indicates persistence length in diglyme between 70 and 81% of that in glycerol (see text and SI Appendix, section 1d).

Fig. 2.

Threading intercalation accelerated by PEG. The complex [μ‐bidppz(phen)₄Ru₂]⁴⁺ (structure in SI Appendix, Fig. S5) is only fluorescent when intercalated in DNA. (A) Representative fluorescence kinetic traces of intercalation in PEG. (B) The major exponential rate constant k (fitting in SI Appendix, section 3) is independent of salt concentration. Salt concentration pooled to estimate SD (error bars ±2 SD, data missing for 40%, 300 mM due to precipitation).

Fig. 3.

Optical tweezers force spectroscopy. DNA stability measured by overstretch force is significantly weakened in 20% diglyme. (Inset) Absence of glyoxal reaction in 20% diglyme proves that loss of DNA stretch stability is not due to local denaturation or increased unpairing breathing. Increased slope of force-elongation curve in the main figure is consistent with decreased unpairing breathing in diglyme.

Fig. 4.

Nanochannel experiments. Extension of λ-DNA with and without 150 mM NaCl versus PEG concentration. DNA confined in 100 × 150 nm² channels. (Inset) A montage of fluorescence images of DNA molecules corresponding to the 4 red points. (Scale bar, 2 µm.) Cartoons show how reduced persistence length also reduces extension.

Fig. 5.

Simplistic models of DNA unstacking: (I) normal B form, (II) slightly extended and unwound B-DNA, (III) inhomogeneously extended B-DNA with holes, and (IV) inhomogeneously extended B-DNA with repeating base triplets and holes [cf. Σ DNA (16)].