posted on 2021-07-26, 15:38authored byAdam S. Backer, Graeme A. King, Andreas S. Biebricher, Jack W. Shepherd, Agnes Noy, Mark C. Leake, Iddo Heller, Gijs J. L. Wuite, Erwin J. G. Peterman
The combination of
DNA force spectroscopy and polarization microscopy
of fluorescent DNA intercalator dyes can provide valuable insights
into the structure of DNA under tension. These techniques have previously
been used to characterize S-DNAan elongated DNA conformation
that forms when DNA overstretches at forces ≥ 65 pN. In this
way, it was deduced that the base pairs of S-DNA are highly inclined,
relative to those in relaxed (B-form) DNA. However, it is unclear
whether and how topological constraints on the DNA may influence the
base-pair inclinations under tension. Here, we apply polarization
microscopy to investigate the impact of DNA pulling geometry, torsional
constraint, and negative supercoiling on the orientations of intercalated
dyes during overstretching. In contrast to earlier predictions, the
pulling geometry (namely, whether the DNA molecule is stretched via opposite strands or the same strand) is found to have
little influence. However, torsional constraint leads to a substantial
reduction in intercalator tilting in overstretched DNA, particularly
in AT-rich sequences. Surprisingly, the extent of intercalator tilting
is similarly reduced when the DNA molecule is negatively supercoiled
up to a critical supercoiling density (corresponding to ∼70%
reduction in the linking number). We attribute these observations
to the presence of P-DNA (an overwound DNA conformation). Our results
suggest that intercalated DNA preferentially flanks regions of P-DNA
rather than those of S-DNA and also substantiate previous suggestions
that P-DNA forms predominantly in AT-rich sequences.