Single Supercoiled DNAs

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Single Supercoiled DNAs
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DNA Supercoiling in vivo

• In most organisms, DNA is negatively supercoiled
• (s -0.06)
• Actively regulated by topoisomerases, ubiquitous
  and essential family of proteins
• Supercoiling is involved in DNA packaging around
  histones, and the initiation of transcription,
  replication, repair recombination
• Known to induce structural changes in DNA
• Traditional means of study (gel electrophoresis,
  sedimentation analysis, cryo-EM) do not provide
  for time-resolved, reversible studies of DNA
  supercoiling

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Topological formalism for torsionally constrained
DNA
Tw (Twist, the number of helical turns of
the DNA) Wr (Writhe, the number of loops
along the DNA) _____ Lk (Total number of
crossings between the 2 strands)
Linking number for torsionally relaxed DNA Lko
Two (Two 1 per 10.5 bp of B-DNA, Wro
0) Linking number for torsionally strained
DNA DLk Lk-Lko DTw Wr Normalized linking
number difference s DLk /Lko
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How to torsionally constrain DNA?
DNA must be 1) unnicked and 2) unable to rotate
at its ends
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Magnetic Trap
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Depth Imaging
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One molecule or two molecules?
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Extension vs. Supercoiling
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(No Transcript)
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Supercoiling and the buckling transition
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Is DNA stretched and supercoiled in vivo or in
solution?

• Relationship between plasmid and extended DNA.

Circular l-DNA with s -0.05 experiences
an internal (entropic) tension 0.3 pN
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Temperature-dependence of DNA helicity
As the temperature increases the DNA helicity
progressively increases (i.e. the angle between
base pairs increases).
Raising the temperature by 15oC causes l-DNA to
unwind by 25 turns
?DNA unwinds by 0.012o/oC/bp
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Force-extension curves for SC-DNA
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Effect of ionic conditions
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Evidence for DNA unwinding hybridization
experiments
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Hybridization force and hat curve detection
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Sequence/Supercoiling dependence of hybridization
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Measuring DNA Unwinding Energeticsusing
low-force data
scDNA
-scDNA
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Paths to Stretched Overwound DNA
TA WAB WAB TB
TA DWAB TB
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Paths to Stretched, Unwound DNA
A- A
DWAB-
TA- DWAB- TB-
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Denaturing DNA before the buckling transition
(2pnc)2 Ed
TB-
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Measuring the Work Deficit to Stretch Unwound DNA
A- A
DWAB-
Symmetry of plectoneme formation TA- TA
D DWAB - DWAB- TB - TB-
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Determination of DNA twist persistence
length,critical torque for unwinding, and
energy of denaturation
kBT C
(2pnc)
-
9 pN nm
Gc
lo
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High-force properties of supercoiled DNA
Negative Supercoiling
Positive Supercoiling
S-DNAP-DNA
S-DNA
Leger et al., PRL (1999) 83 1066-1069
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DNA the compliant polymorph
B-DNA 10.4 bp/turn 3.3 nm pitch
P-DNA 2.5 bp/turn 1.5nm/bp
S-DNA 38 bp/turn 22 nm pitch
Images R. Lavery using JUMNA
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Effect of torque on transition rates
a aoexp(2pDnnativeG/kBT)
b bo exp(-2pDnunwoundG/kBT)