Single Supercoiled DNAs
description
Transcript of Single Supercoiled DNAs
![Page 1: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/1.jpg)
Single Supercoiled DNAs
![Page 2: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/2.jpg)
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
![Page 3: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/3.jpg)
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
![Page 4: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/4.jpg)
How to torsionally constrain DNA?
DNA must be 1) unnicked and 2) unable to rotate at its ends
![Page 5: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/5.jpg)
Magnetic Trap
![Page 6: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/6.jpg)
Depth Imaging
![Page 7: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/7.jpg)
One molecule or two molecules?
![Page 8: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/8.jpg)
Extension vs. Supercoiling
![Page 9: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/9.jpg)
![Page 10: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/10.jpg)
Supercoiling and the buckling transition
![Page 11: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/11.jpg)
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
![Page 12: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/12.jpg)
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
![Page 13: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/13.jpg)
Force-extension curves for SC-DNA
![Page 14: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/14.jpg)
Effect of ionic conditions
![Page 15: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/15.jpg)
Evidence for DNA unwinding: hybridization experiments
3
![Page 16: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/16.jpg)
Hybridization : force and hat curve detection
![Page 17: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/17.jpg)
Sequence/Supercoiling dependence of hybridization
![Page 18: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/18.jpg)
Measuring DNA Unwinding Energeticsusing low-force data
-scDNA
+scDNA
![Page 19: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/19.jpg)
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
![Page 20: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/20.jpg)
Paths to Stretched, Unwound DNA
A A- B- = A B B-twist stretch stretch twist
TA- + WAB- = TB-
A- = A+
WAB-
![Page 21: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/21.jpg)
Denaturing DNA before the buckling transition
(2nc)2 + Ed12
kBT Clo
TB- =
=kBT C
lo(2n)
Ed= 2(n-nc)c-
![Page 22: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/22.jpg)
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
![Page 23: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/23.jpg)
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 n
m
)
![Page 24: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/24.jpg)
High-force properties of supercoiled DNA
Leger et al., PRL (1999) 83: 1066-1069
Negative Supercoiling Positive Supercoiling
S-DNA
S-DNA+P-DNA
![Page 25: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/25.jpg)
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
![Page 26: Single Supercoiled DNAs](https://reader036.fdocuments.in/reader036/viewer/2022062501/56815e07550346895dcc5bdc/html5/thumbnails/26.jpg)
Effect of torque on transition rates
= oexp(2nnative/kBT) = o exp(-2nunwound/kBT)