Andresen, Kurt

Bull, Abby

Meng, Wei

Qiu, Xiangyun

Elsevier - Publisher Connector 

Ion Binding and Electrostatics in Condensed DNA Arrays Probed by Ion Counting Techniques 

Monday, February 9, 2015 233asuch as DNA. Traditional magnetic tweezers geometry imposes a vertical
extension on a surface-tethered molecule parallel to the microscope’s optic
axis, resulting in only a small section of DNA being in focus and thus being
reliant on indirect measurements of magnetic bead position to infer topological
dynamics. Here we describe a bespoke transverse magnetic tweezers system
that can be integrated with optical tweezers and multicolor single-molecule
fluorescence imaging. This permits independent control over the molecular
extension and torque imposed on a single DNA molecule while allowing the
full extent of the DNA to be visualized within the fluorescence microscope’s
depth-of-field, in addition to proteins bound to the DNA which affect its topol-
ogy, such as DNA gyrase. Using a lamda DNA control we have generated a
bifunctionalized construct via oligo insertion to permit tethering of single
DNA molecules between a streptavidin-coated paramagnetic bead and a nano-
scale platform, and have generated superresolution structural detail of DNA
with video-rate sampling to a lateral precision of a few tens of nm utilizing
the stochastic photoblinking of the intercalating DNA dye YOYO-1 and the
minor groove binding dye SYTO-13 through Blinking assisted Localization
Microscopy (BaLM).
1171-Pos Board B122
Tension-Dependent Stability of Torsionally Constrained DNA: Melting
Precedes Overwinding
Graeme A. King, Erwin J.G. Peterman, Gijs J.L. Wuite.
Department of Physics and Astronomy, VU University Amsterdam,
Amsterdam, Netherlands.
Key genomic processes such as replication and transcription require localized
unwinding and base-pair melting of the double-helix. In vivo, however, DNA is
often torsionally constrained, presenting a topological barrier to unwinding.
While it is known that unwinding can proceed through buckling of such
DNA (resulting in plectonemes), this may only occur at low forces (typically
below 1 pN). Here, we ask: how can torsionally constrained DNA unwind at
higher forces without changing the overall twist?
Using a combination of optical tweezers and fluorescence microscopy we map
the local changes in twist and base-pair integrity of torsionally constrained
DNA as a function of tension and ionic strength. By correlating the second de-
rivatives of force-extension curves of torsionally constrained DNA to the
binding of a fluorescently-labeled single-stranded DNA binding protein (Repli-
cation Protein A), we identify two tension-dependent structural transitions. In
agreement with a previous proposal, we show that at high tension (above
115 pN), unwinding is accompanied by the formation of overwound DNA
(termed P-DNA) in a largely cooperative mechanism. Strikingly, we also reveal
that for intermediate tensions (60 to 115 pN), localized base-pair melting can
occur without changes in DNA twist. This mechanism, which is significantly
less cooperative, is referred to here as ‘pre-melting’. We demonstrate that
pre-melting is favored by AT-rich sequences and low ionic strength. In
contrast, P-DNA is largely stabilized by higher salt concentrations, while
also having a preference for AT-rich domains. This supports the hypothesis
that P-DNA consists of tightly entwined backbones with exposed bases.
These findings provide a new understanding of the interplay between DNA
twist, extension and sequence. Moreover, since DNA unwinding can be
induced through either tension or applied torque, this work may have strong im-
plications for the metabolism of torsionally constrained DNA in vivo.
1172-Pos Board B123
Estimating Persistence Length of DNA from Molecular Dynamics
Simulations
Aleksander V. Drozdetski, Ramu Anandakrishnan, Alexey Onufriev.
Virginia Tech, Blacksburg, VA, USA.
Bending rigidity of DNA is one of its most important mechanical properties,
since in eukaryotic cells a vast majority of the DNA (75-80%) is tightly packed
into chromatin. Classical experiments have measured the persistence length
(PL) of DNA, which is directly related to its bending rigidity, to be about
45-50 nm (140-160 bp). Previous all-atom molecular dynamics (MD) studies
of DNA flexibility attempted to calculate this value using relatively small frag-
ments (much less than PL) in explicit solvent. However, the simulations are
usually computationally expensive due to a large number of water molecules
required to construct the solvent box, and the resulting PL approximations
are greater than the experimental values. One possible explanation for this
discrepancy between experiment and simulation may be that classical experi-
mental measurements were performed with DNA fragments on the order of
PL or greater, while fragments much smaller than PL have so far been used
in MD studies. In this work we provide a computationally inexpensive method
of estimating the PL of DNA from atomistic MD simulations of long fragments
on the order of PL (~150 bp) in implicit solvent that provides good agreement
with experiment.1173-Pos Board B124
Dual-Beam Fluorescence Correlation Spectroscopy of DNA Hairpin
Folding-Unfolding Reaction at Various Temperatures and Salt
Concentrations
Farshad Abdollah-Nia1, Martin Gelfand1, Alan Van Orden2.
1Physics, Colorado State University, Fort Collins, CO, USA, 2Chemistry,
Colorado State University, Fort Collins, CO, USA.
Fluorescence Correlation Spectroscopy has been used to study the conforma-
tional dynamics of DNA hairpin molecules of various loop lengths in aqueous
solution. In a recent development, we are also able to monitor the temperature
dependence of the processes of interest. In our 2-beam approach, the analyte
molecules flow through an electrophoresis capillary under the influence of pres-
sure driven flow or electrophoretic flow and are probed by two spatially offset
probe volumes in such a way that the molecules flow sequentially from one
probe volume to the next. A pair of dye-quencher molecules attached to
DNA or RNA hairpin structures give rise to fluorescence fluctuations due to
folding-unfolding dynamics of the hairpins. Fluorescence fluctuations from
the probe volumes are analyzed using auto-correlation and cross-correlation
analysis. In this way, we are able to resolve fluorescence fluctuation time con-
stants arising from diffusion, flow, triplet blinking, and conformational fluctu-
ations. Using a temperature controlling stage we can fix the temperature to
within 0.2 degrees in the range 5 - 95 degrees Celsius, unaffected by the
flow rate of the solution in the capillary. In this presentation, we will discuss
recent results from our study of DNA hairpins of different loop lengths, at
different temperatures and Sodium ion concentrations. We will also discuss
binding and unbinding of counterions to individual nucleotides as they flow
through the capillary under the influence of an applied electric field. Emphasis
will be placed on how the desired information can be extracted using our unique
approach to fluorescence correlation spectroscopy.
1174-Pos Board B125
Ion Binding and Electrostatics in Condensed DNA Arrays Probed by Ion
Counting Techniques
Kurt Andresen1, Wei Meng2, Abby Bull1, Xiangyun Qiu2.
1Department of Physics, Gettysburg College, Gettysburg, PA, USA,
2Department of Physics, The George Washington University, Washington,
DC, USA.
Despite decades of theoretical and experimental research, the mechanism of
DNA condensation remains elusive. Numerous models successfully describe
this condendsation, including those invoking dynamic counterion fluctuations,
salt bridges, static counterion lattices, and models that invoke the underlying
DNA geometry, all while presenting vastly different models for ion binding.
Here we present new experimental data that quantifies the ions bound to
condensed DNA arrays. By measuring the competition between condensing
ions and various non-condensing ions we quantify the electrostatic interactions
between the DNA and ions. Furthermore, by varying the inter-axial spacing of
condensed DNA utilizing crowding agents, we report on the interplay between
the packing force of DNA and competitive ion binding. Finally, we present a
simple new Ion Binding Model (IBM) that captures much of the observed bind-
ing. These data should prove vital in determining the underlying electrostatic
mechanisms that drive DNA condensation.
1175-Pos Board B126
Brownian Dynamics Study of DNA Supercoil Relaxation
Ikenna D. Ivenso, Todd D. Lillian.
Mechanical Engineering, Texas Tech University, Lubbock, TX, USA.
The stresses induced in DNA during cell processes such as replication and tran-
scription lead to the formation of plectonemic supercoils. Supercoiling in turn
affects these processes. As a result of this relationship between supercoiling and
these processes, the degree of supercoiling in DNA needs to be controlled
closely in order to optimize these cell processes. This control is facilitated by
enzymes such as type 1b topoisomerases and nicking endonucleases which
relax supercoiling in DNA.
The dynamics of DNA supercoil relaxation have been studied in recent exper-
iments by means of single-molecule magnetic and/or optical tweezer ex-
periments. Novel as these experiments are, they do not permit a direct
observation of the structural changes that occur in DNA during supercoil relax-
ation. They depend on components (e.g. a paramagnetic bead) attached to the
DNA to indirectly obtain information about these dynamics.
We studied the dynamics of supercoil relaxation by means of Brownian Dy-
namics simulations of a discrete wormlike-chain (dWLC) model of DNA.
These simulations parallel the single-molecule experiments in which a single
DNA molecule is held under constant tension so that its end-to-end extension
increases as supercoils are relaxed by a nicking endonuclease. The dWLC
model accounts for elasticity, electrostatics and entropic forces as well as for
hydrodynamic interactions.


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Ion Binding and Electrostatics in Condensed DNA Arrays Probed by Ion Counting Techniques

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