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Single Supercoiled DNAs

Single Supercoiled DNAs. DNA Supercoiling in vivo. In most organisms, DNA is negatively supercoiled ( s ~ -0.06) Actively regulated by topoisomerases , ubiquitous and essential family of proteins

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Single Supercoiled DNAs

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  1. Single Supercoiled DNAs

  2. 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

  3. 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

  4. How to torsionally constrain DNA? DNA must be 1) unnicked and 2) unable to rotate at its ends

  5. Magnetic Trap

  6. Depth Imaging

  7. One molecule or two molecules?

  8. Extension vs. Supercoiling

  9. Supercoiling and the buckling transition

  10. 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

  11. 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

  12. Force-extension curves for SC-DNA

  13. Effect of ionic conditions

  14. Evidence for DNA unwinding: hybridization experiments 3

  15. Hybridization : force and hat curve detection

  16. Sequence/Supercoiling dependence of hybridization

  17. Measuring DNA Unwinding Energeticsusing low-force data +scDNA -scDNA

  18. twist stretch stretch twist A  A+  B+ = A  B B+ 1 kBT C (2pn)2 lo 2 Paths to Stretched & Overwound DNA TA+ + WA+B+ = WAB + TB+ TA+ + DWAB+ = TB+ =

  19. twist stretch stretch twist A  A-  B- = A  B B- Paths to Stretched, Unwound DNA A- = A+ DWAB- TA- + DWAB- = TB-

  20. kBT C (2pn) G = lo 1 kBT C - Ed= 2p(n-nc)Gc lo 2 Denaturing DNA before the buckling transition (2pnc)2 + Ed TB- =

  21. 2p2 kBT C (n-nc)2 lo Measuring the Work Deficit to Stretch Unwound DNA A- = A+ DWAB- Symmetry of plectoneme formation: TA- = TA+ D = DWAB+ - DWAB- = TB+ - TB- =

  22. 1/2 (in nm ) Determination of DNA twist persistence length,critical torque for unwinding, and energy of denaturation kBT C - (2pnc) ~ 9 pN nm Gc= lo

  23. High-force properties of supercoiled DNA Negative Supercoiling Positive Supercoiling S-DNA+P-DNA S-DNA Leger et al., PRL (1999) 83: 1066-1069

  24. 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

  25. Effect of torque on transition rates a = aoexp(2pDnnativeG/kBT) b = boexp(-2pDnunwoundG/kBT)

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