DNA A-tracts Are Not Curved in Solutions Containing High Concentrations of Monovalent Cations

Abstract

The intrinsic curvature of seven 98-base pair DNA molecules containing up to four centrally located A6-tracts has been measured by gel and capillary electrophoresis as a function of the number and arrangement of the A-tracts. At low cation concentrations, the electrophoretic mobility observed in polyacrylamide gels and in free solution decreases progressively with the increasing number of phased A-tracts, as expected for DNA molecules with increasingly curved backbone structures. Anomalously slow electrophoretic mobilities are also observed for DNA molecules containing two pairs of phased A-tracts that are out of phase with each other, suggesting that out-of-phase distortions of the helix backbone do not cancel each other out. The mobility decreases observed for the A-tract samples are due to curvature, not cation binding in the A-tract minor groove, because identical free solution mobilities are observed for a molecule with four out-of-phase A-tracts and one with no A-tracts. Surprisingly, the curvature of DNA A-tracts is gradually lost when the monovalent cation concentration is increased to ~200 mM, regardless of whether the cation is a hydrophilic ion like Na+, NH4+ or Tris+ or a hydrophobic ion like tetrabutylammonium (TBA+). The decrease of A-tract curvature with increasing ionic strength, along with the known decrease of A-tract curvature with increasing temperature, suggests that DNA A-tracts are not significantly curved under physiological conditions.

Flanking A·T Basepairs Destabilize the B∗ Conformation of DNA A-Tracts

“…Three observations suggest that the mobility differences are not due to differences in shape. First, differences in shape are not observed in BGEs containing 300 mM cation (19). Second, previous studies have shown that the translational diffusion constants of small DNA Biophysical Journal 108(9) 2291-2299 oligomers containing the same number of basepairs are independent of the presence or absence of A-tracts, within experimental error (43,45).…”

“…We have also characterized the dependence of DNA electrophoretic mobilities on the ionic strength of the solution and the charge density of the polyion (49)(50)(51). Finally, we showed that the curvature of A-tract-containing DNA molecules is markedly reduced in solutions containing~200 mM cation, even though monovalent cations are still preferentially localized in the A-tract minor groove (19). Hence, curvature of the DNA helix backbone and the preferential localization of monovalent cations in the A-tract minor groove are not necessarily linked.…”

“…The midpoint of the B* 4 B transition occurs at temperatures between 30 and 40 C, depending on the A-tract sequence and the ionic strength of the solution (13)(14)(15)(16)(17)(18). Furthermore, the macroscopic curvature of the DNA backbone caused by A-tract phasing is nearly eliminated in solutions containing~200 mM monovalent cations (19).…”

“…Here, we use free solution capillary electrophoresis (CE) to analyze the effect of the flanking sequences on the interaction of monovalent cations with DNA A-tracts. CE is a useful technique for such studies because the electrophoretic mobility of a small DNA oligomer is directly proportional to its effective charge after counterion condensation (38)(39)(40)(41)(42) and inversely proportional to its translational friction coefficient (19,43,44). Because the translational diffusion coefficients of small DNA oligomers containing the same number of basepairs are essentially independent of the presence or absence of A-tracts (42,43,45), the free solution mobilities of small DNA oligomers reflect differences in effective charge due to sequence-dependent cation interactions.…”

In vitro selection of DNAs with an increased propensity to form small circles

“…Such bends are dynamic and generally quite gradual, covering several helical turns . It has long been known that both the direction and extent of DNA bending depends upon its electrostatic charge and sequence due to variations in stacking between adjacent base pairs . In general, a pyrimidine followed by a purine is more deformable than other dinucleotides, and dinucleotides comprised of adenines and thymines are more anisotropically flexible than those comprised of cytosines and guanines .…”