1. DNA: Structure, Dynamics and Recognition Les Houches 2004 L4: DNA deformation

2. BASE PAIR OPENING

3. Biological time scale Bond vibrations1 fs(10 -15 s) Sugar repuckering1 ps(10 -12 s) DNA bending 1 ns(10 -9 s) Domain movement1  s(10 -6 s) Base pair opening1 ms(10 -3 s) Transcription2.5 ms / nucleotide Protein synthesis6.5 ms / amino acid Protein folding~ 10 s RNA lifetime~ 300 s

4. Enzymatic base chemistry

5. Adenine-Thymine base pair HN3 imino proton S S

6. Guanine-Cytosine base pair HN1 imino proton S S

7. Base opening lifetimes GC15-25 ms AT 5-10 ms C G C A A G A A G C G * * 4 1 1 23 4 5 4 * *

8. A 4 T 4 versus T 4 A 4 T T T T A A A A 1 17 19 4 4 19 17 1 A A A A T T T T 60 100 100 65 65 100 100 60 Leroy et al. Biochemistry 27, 1988, 8894 Base pair lifetimes (ms) 15°C

9. B-DNA - 2ns dynamic trajectory

10. Free energy calculations using restrained opening Guidice et al. ChemPhysChem 2, 2001, 673 Varnai & Lavery J. Am. Chem. Soc. 124, 2002, 7272

11. WHAM FREE ENERGY PROFILE BIASED PROBABILITY HISTOGRAM N(q) W(q) Reaction coordinate (q) Nw  P* i (q) exp [  V i (q)] i =1 P i (q)   Nw  n i exp  [F i (q)  V i (q)] i =1 Nw F i (q)   kT ln  P i (q) i =1

12. B-DNA oligonucleotide studied CTCTCTCTCTCTC GAGAGAGAGAGAG

13. Extraction d’une base de l’ADN

14. Closed AT pair

15. Adenine –50° (minor)

16. Adenine –100° (minor)

17. Adenine +50° (major)

18. Adenine +100° (major)

19. Free energy curves for base opening

20. Imino proton accessibility (Å 2 ) T G

21. Base movements are coupled  Adenine (°)  Thymine (°)  Adenine (°)

22. Sequence effects on opening: A-tracts  T A-tract T Ref

23. Bending amplitude (°)  < -50° -50° <  < +50°  > +50° G T

24. A word of warning!

25. BASE FLIPPING

26. Hha1 methyltransferase Klimašauskas et al. Cell 76 (1994) 357

27. Minor groove  Major groove

28. -200° opening +160° opening

29. Backbone rearrangements

31. SUPERCOILING

32. DNA supercoiling (circular plasmid)

33. DNA supercoiling L = linking number = number of strand crossings T = twist = number of turns of double helix W = writhe = number of helix crossovers L = T + W  = supercoiling density = (L – L 0 ) / L 0 =  L / L 0 typically  ~ -0.06 (1 crossing less per 17 turns)

34. Linking number (L or L k ) – a topological constant

35. Twist (T) versus Writhe (W) Low forceHigh force

36. L = T + W

37. Interwound and toroidal forms of a negatively supercoiled plasmid L.H. R.H.

38. Ethidium bromide intercalates into DNA and reduces its twist by ~26°

39. Effect of an intercalator on a negatively supercoiled plasmid

40. Topoisomerases Topoisomerase I- single strand cuts - releases negative supercoiling Topoisomerase II- double strand cuts (eukaryotes)- releases negative supercoiling Topo II (gyrase)- generates negative supercoiling (prokaryotes)- consumes ATP Reverse gyrase- generates positive supercoiling (thermophiles)

41. Topoisomerase I – single strand cuts

42. Topoisomerase II – double strand cuts

43. Topo II (gyrase) DNA wrapping

44. DNA packed on nucleosomes

45. Nucleosome – schematic view

46. EXTREME DEFORMATIONS

47. DNA stretching Cluzel et al. Science 271, 1996, 792

48. 70 pN phase transition

49. S-DNA: fibre and ribbon forms

50. Fibre diffraction of stretched DNA Greenall et al. J. Mol. Biol. 2001, 305, 669 Rise ~ 5.6 Å Helix spacing ~ 13 Å

51. TBP-DNA complex

52. DNA: local stretching 3' 5' 3' 5' Major Minor

53. DNA: global and local 3'3' stretching

54. TBP induced deformation X-rayModel

55. Magnetic twisting control DIG : AntiDIG Biotin : Streptavidin NS Strick et al. Biophys. J. 74, 1998, 2016 Allemand et al. Proc. Natl. Acad. Sci. (USA) 95, 1998, 14152

56. Twisted DNA forms plectonemes

57. DNA twisting under tension > 3 pN < 0.3 pN

58. Simulation of DNA twisting

59. Simulating twisting