Relative Contributions of Base Stacking and Electrostatic Repulsion on DNA Nicks and Gaps

In
duplex DNA, the continuous sugar phosphate backbones prevent
the double helix from significant bending, but breaks in the duplex
such as nicks, gaps, and flaps present points at which significant
bending is possible. The conformational dynamics of these aberrant
structures remains poorly understood. Two factors can maintain the
duplexlike conformation of these aberrant structures, these being
the hydrophobic and aromatic stacking interactions of the nucleobases,
and the electrostatic repulsion of the negatively charged backbones.
Using confocal single-molecule Förster resonance energy transfer
on nicked and gapped DNA structures, we compare the relative contributions
of these two factors by modulating the electrostatic repulsion through
mono- and divalent cation concentrations. Base stacking interactions
dominate the dynamics of nicked DNA, making it behave essentially
like duplex DNA. Gapped structures have weaker base stacking and thus
backbone electrostatic repulsion becomes important, and shielding
from cations results in an average increase in bending around the
gap. This bending of gapped structures could be interpreted by increased
flexibility of unstacked structures, transient unstacking events,
or a combination of the two. Burst variance analysis (BVA) and analysis
by photon-by-photon hidden Markov modeling (H2MM), methods
capable of detecting submillisecond dynamics of single molecules in
solution, only revealed a single state, indicating that dynamics are
occurring at time scales shorter than microseconds.