single supercoiled dnas. dna supercoiling in vivo in most organisms, dna is negatively supercoiled (...
TRANSCRIPT
Single Supercoiled DNAs
DNA Supercoiling in vivo
• In most organisms, DNA is negatively supercoiled (~ -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
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
Lk = Lk-Lko = Tw + Wr
Normalized linking number difference
= Lk /Lko
How to torsionally constrain DNA?
DNA must be 1) unnicked and 2) unable to rotate at its ends
Magnetic Trap
Depth Imaging
One molecule or two molecules?
Extension vs. Supercoiling
Supercoiling and the buckling transition
Is DNA stretched and supercoiled in vivo or in solution?
• Relationship between plasmid and extended DNA.
Circular-DNA with ~ -0.05
experiences aninternal (entropic)tension ~ 0.3 pN
Temperature-dependence of DNA helicity
As the temperature increases the DNA helicity progressivelyincreases (i.e. the angle between base pairs increases).
Raising the temperature by 15oC causes -DNA to unwind by ~ 25 turns
DNA unwinds by ~ 0.012o/oC/bp
Force-extension curves for SC-DNA
Effect of ionic conditions
Evidence for DNA unwinding: hybridization experiments
3
Hybridization : force and hat curve detection
Sequence/Supercoiling dependence of hybridization
Measuring DNA Unwinding Energeticsusing low-force data
-scDNA
+scDNA
Paths to Stretched & Overwound DNA
A A+ B+ = A B B+twist stretch stretch twist
TA+ + WA+B+ = WAB + TB+
TA+ + WAB+ = TB+ = (2n)212
kBT Clo
Paths to Stretched, Unwound DNA
A A- B- = A B B-twist stretch stretch twist
TA- + WAB- = TB-
A- = A+
WAB-
Denaturing DNA before the buckling transition
(2nc)2 + Ed12
kBT Clo
TB- =
=kBT C
lo(2n)
Ed= 2(n-nc)c-
Measuring the Work Deficit to Stretch Unwound DNA
A- = A+
WAB-
Symmetry of plectoneme formation: TA- = TA+
= WAB+ - WAB- = TB+ - TB- = 22 kBT C
lo(n-nc)2
Determination of DNA twist persistence length,critical torque for unwinding, and energy of denaturation
c=kBT C
lo(2nc)- ~ 9 pN nm
1/2
(in
nm
)
High-force properties of supercoiled DNA
Leger et al., PRL (1999) 83: 1066-1069
Negative Supercoiling Positive Supercoiling
S-DNA
S-DNA+P-DNA
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
Effect of torque on transition rates
= oexp(2nnative/kBT) = o exp(-2nunwound/kBT)