Treball presentat a l'assignatura de Deontologia i Veterinària Legal (21223

Doye, Jonathan P.K.

Harrison, Ryan M.

Louis, A.A.

Biophysical Journal

Harrison, RM

Louis, Adriaan

Doye, Jonathan

ORA - Oxford University Research Archive

Coarse-Grained Modelling of Extreme DNA Bending

Trabal Garmon, Alba.

Dordas-Perpinyà, Marta

Diposit Digital de Documents de la UAB

Apicultura ecològica vs. tradicional

Louis, AA

Doye, JPK

Oxford University Research Archive (ORA)

Elsevier - Publisher Connector 

66a Sunday, February 16, 2014In addition, the temperature dependence of the transition rates allowed us to
determine the energy barrier between the states, which was ~ 12 kcal/mol. In
comparison with a guanine-cytosine (GC) repeat sequence, a TG repeat
sequence requires higher torsional stress to trigger the B-Z transition but has
a significantly lower energy barrier under conditions the B- and Z-DNA states
are equally populated. As a dynamic mechanical sensor, TG repeat sequences
can function as a regulatory unit controlling global mechanics of DNA.
355-Pos Board B110
Coarse-Grained Modelling of Extreme DNA Bending
Ryan M. Harrison1, A.A. Louis2, Jonathan P.K. Doye1.
1Physical & Theoretical Chemistry Laboratory, University of Oxford,
Oxford, United Kingdom, 2Rudolf Peierls Centre for Theoretical Physics,
University of Oxford, Oxford, United Kingdom.
There is interest in the nature of extreme bending in duplex DNA, due to its
critical biological role in genome regulation and more fundamental structural
and thermodynamic implications. The worm-like chain (WLC) model success-
fully predicts the behavior of DNA at moderate curvatures; however, at larger
curvatures, there is a crossover from the WLC regime, where the stress is uni-
formly distributed through the helix, to an extreme bending regime, where the
stress is localized at a ‘‘defect’’. Questions remain about the microscopic nature
of extreme bending, specifically the presence of local deformations, their struc-
tures and related thermodynamic costs.
We address these issues using oxDNA, a coarse-grained model of DNA that has
provided insight into a diverse array of DNA systems, from the fundamentals of
hybridization to applied work on DNA nanomachines and DNA origami.
oxDNA reproduces results for cyclization
equilibria and DNA duplexes bent due to
internal strains, reported through differen-
tial melting and a FRET-based approach.
Furthermore, the simulations directly
probe the thermodynamic and structural
properties of extreme bending in these systems, in particular, finding that the
bending is localized at a kink where one or two base pairs have melted (Figure,
arrow).
356-Pos Board B111
Transient Kinetics Measured with Force Steps Discriminate between
Double Stranded DNA Elongation and Melting and Define the Reaction
Energetics
Pasquale Bianco, Lorenzo Bongini, Luca Melli, Vincenzo Lombardi.
Department of Evolutionary Biology, University of Florence, Sesto
Fiorentino, Italy.
Double stranded DNA pulled to ~65 pN undergoes an overstretching transition
from the basic conformation (B-form) to a 1.7 times longer conformation that
represents a fundamental stage in the structural transitions during DNA recom-
bination, replication and repair. By using a dual laser optical tweezers with a
fast force feedback (2 ms rise time), we recorded the length transient following
force steps imposed on the l-phage DNA with different degrees of melting and
at different temperatures (10-25C). The rate-force relations obtained from the
lengthening transient under our rapid force clamp following 2-35 pN force pull
to the overstretching force shows that the whole 70% extension is a two state
reaction from the B-form to an S-form that precedes and is independent of
melting. The shortening transient following 20-35 pN force drop from the over-
stretching force is made by stepwise shortenings and pauses due to S-B transi-
tions and reannealing of the melted segments. The temperature dependence of
the lengthening transient shows that the entropic contribution to the reaction at
room temperature is only 1/3 of the entropy change expected from thermal
melting. The structural parameters that describe the distances from the B and
the S state to the transition state, do not depend on temperature. Accordingly
the total elongation of a single two-state unit, and therefore q, the index of
cooperativity (22bp) of the reaction, do not depend on temperature, suggesting
that it arises from structural factors, such as the nucleic acid sequence.
Supported by IIT-SEED (Genova) and Ente Cassa di Risparmio di Firenze.
357-Pos Board B112
Diaminopurine-Substitution Modify DNA Elasticity and Favors L-Helices
Qing Shao1, Monica Fernandez2, Sharon Owino3, Yoojin Lee2, Laura Finzi2,
David Dunlap3.
1School of Science and Technology, Georgia Gwinnett College,
Lawrencevill, GA, USA, 2Department of Physics, Emory University, Atlanta,
GA, USA, 3Department of cell Biology, Emory University, Atlanta, GA,
USA.Proteins searching DNA for recognition sites in order to carry out replication,
transcription, or mitosis must navigate on this remarkably stiff polymer. Elec-
trostatics, base stacking and hydrogen bonding all contribute to stiffness and
presumably limit deformability. Therefore, recognition depends not only on
sequence per se but also on the stiffness and conformational dynamics of a
sequence. Single molecule experiments have demonstrated a facile, progres-
sive transition from a B- to an L-form helix that begins at negative linking
numbers of a few percent when DNA gently stretched by as little as one pi-
coNewton is progressively uncoiled. Since the particular sequence of a DNA
segment heavily influences both base stacking and hydrogen bonding, the
conformational dynamics of B-to-L transitions should be tunable. As a funda-
mental test of this, the DNA was synthesized by PCR substituting adenine
with diaminopurine to establish triple hydrogen bonding for all base pairs.
Triple hydrogen bonding stiffened the DNA by 30% flexurally, perhaps
due to reduced helix breathing and enhanced base stacking, while softened
DNA by 30% torsionally. Compared to normal DNA, upon unwinding under
high tension (> 1 pN) DAP-substituted DNA more readily transitioned from
B to L. Furthermore, DAP-substituted DNA formed plectonemes with larger
gyres for both B- and L-form helices. This model of enhanced hydrogen
bonding and base stacking achieved with DAP substitution indicates that
the L-form helix forms spontaneously with high frequency under physiolog-
ical conditions.
358-Pos Board B113
Biochemical and Biophysical Properties of Positively Supercoiled DNA
Andrea M. Berrido, Andrew Chen, Yuk-Ching Tse-Dinh, Fenfei Leng.
Chemistry & Biochemistry, Florida International Univ, Miami, FL, USA.
Here we studied the biophysical and biochemical properties of the positively
supercoiled DNA under different conditions. Large amount of positively
supercoiled DNA is difficult to obtain compared to the natural-occurring
negatively supercoiled DNA. Therefore, the biophysical and biochemical
properties have not been fully explored. In this report we utilized Archaeo-
globus fulgidus DNA reverse gyrase, which was expressed in E. coli strain
BL21(DE3)/pLysS and purified using an optimized purification scheme, to
convert the negatively supercoilied plasmid DNA to the positively supercoiled
DNA. The positively supercoiled DNA was further purified by the CsCl-EB
density-gradient centrifugation. After we obtained a few milligrams of the
positively supercoiled DNA template, biochemical and biophysical properties
were characterized using different techniques, such as UV melting, circular
dichroism, AFM (atomic force microscope), fluorescence spectrometry,
DNA topoisomerase assays, and etc. Our results are expected to provide the
basis for the utilization of the (þ) supercoiled DNA for future biomedical
applications, such as the use of (þ) supercoiled DNA templates for the
screening of anti-gyrase antibiotics.
359-Pos Board B114
A Combined NMR and Molecular Dynamics Investigation of Sequence
Context Effects on Backbone Dynamics of DNA
Kiley Lawrence.
Scripps College, Claremont, CA, USA.
The mechanisms by which proteins recognize and bind to specific DNA se-
quences underlie the regulation of basic cell cycle events. Protein recognition
sites are frequently regarded in terms of direct readout: as short, rigid sequences
that a protein recognizes via base edge contacts. Protein-DNA recognition often
operates by indirect readout, in which protein-DNA complexes involve con-
tacts to the phosphate backbone. Dynamic variation of these backbone sites
likely plays a large role in binding specificity; one form of dynamic backbone
variation is the conformational equilibrium between the BI and BII substates of
the canonical B-form of DNA.
In this work we perform MD simulations of six DNA sequences, previously
characterized using 31P NMR, that contain biologically relevant protein
recognition sites with differing flanking sequences. The synergistic use of
NMR experiments and MD simulations allows for the rigorous assessment
of BI/BII profiles in order to study sequence context effects on the conforma-
tional ensemble and dynamics of the DNA backbone. Taken together, our
NMR and MD data indicate that sequence context plays a significant role
in backbone dynamics; most prominently, A- and T-heavy flanking sequences
appear to largely quench backbone dynamics. These data also demonstrate
that recent MD force field enhancements are able to successfully reproduce
patterns of backbone structure determined by NMR. Moreover, the MD sim-
ulations enable investigation of sequence steps that remain unresolved in
crowded NMR regions, while the NMR data suggest avenues for future force
field improvements.


Coarse-Grained Modelling of Extreme DNA Bending 

Oxford University Research Archive

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Apicultura ecològica vs. tradicional

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