1. Institute of Physics Chinese Academy of Sciences Beijing, China Ming Li ( 李 明 ) mingli@iphy.ac.cn A single molecule study on the mechanism of UvrD helicase

2. People are used to thinking about biological problems in a single molecular way. From DNA, via RNA, to protein

3. Magnetic tweezers

4. Genes are duplicated before cell division

5. The 2 strands of a DNA must be separated in order for the genes to be duplicated.

6. The machine To CCD

7. Connecting DNA to a surface and a handle Biotin ended digoxigenin T4 ligase is used to connect

8. Force measurement F=2.0 pN F=13.0 pN F mag xx Over damped pendulum

9. DNA follows the WLC model

10. Twisting DNA

13. It is a crucial to DNA damage repair. E. Coli UvrD is a SF1 DNA helicase… helicase

14. and mismatch repair.

15. Cell

17. Nature Reviews

18. Dimer or monomer? The mechanism?

19. Experimental design

20. Binding UnwindingRezipping Expected observations handle hairpin M-bead magnet

21. unwinding-rezipping events

22. Unwinding rate versus force F=5 pN F=9 pN F=5 pN F=9 pN Force hinders UvrD, rather than helps it.

24. A force higher than ~14 pN unzips DNA Force destabilizes DNA

27. A different mechanism for UvrD

28. 1) Dimer is the functional form of UvrD, although UvrDs exists in solution as monomers. [UvrD]=5 nM and 10 nM [ATP]=1 mM

29. 15 nt A loading tail longer than 15 nt is required!

30. 2) There are two binding events before dimerization occurs at the DNA junction

31. Binding kinetics

33. K 1 =0.23 ±0.05 /s; K 2 =0.38 ±0.08 /s @ [UvrD]=5 nM

34. K 1 =0.05 /s; K 2 =0.07 /s @ [UvrD]=1 nM 1/K 1 =20 Sec; 1/K 2 =14 Sec K -1 =0.12 /s @ [UvrD]=1 nM 1/K -1 =8.3 Sec

35. Two binding events at the DNA junction 3’5’ 3’5’ 3’5’ loading unwinding stickingdimerizing

36. 3) Dimerization process is dynamical, assembling and disassembling momently.

37. Details of the unwinding events

38. UW=unwinding; SRW=slow rewinding; FRW=fast rewinding; P=pausing; UB=unbinding UB Details of the unwinding events

39. 3’ 5’ 3’ 5’ loading binding unwinding unbinding

40. 3’ 5’ 3’ 5’ loading binding unwinding rewinding

41. 3’ 5’ 3’ 5’ binding unwinding pausing unbinding

42. 3’ 5’3’ 5’ 3’ 5’ binding unwinding slow rewinding fast rewinding

43. 4) Dimer undergoes a conformational change to become active.

44. Configurational change of the dimer bends the ssDNA tail. Force performs negative work!

45. Configurational change of the dimer bends the ssDNA tail. Force performs negative work!

46. Docking of two UvrDs supports the mechanism. Structures were from the PDB

47. d~0.7 nm v=v 0 exp(-F*d/k B T) v 0 =68 bp/s; JMB(2003) d=0.7 nm Configurational change bends the ssDNA tail by ~50deg.

48. Biological significance A road cleaner!

49. ! Autoinhibitory 2B domain must be released to activate the helicase.

50. Summary EMBO Journal 2008, 27, 3279 Sun et al.

51. Challenge: Can we actually see the details? Improve the machine to get sub-nanometer precision! TIRM+MT

52. Acknowledgement Thank you! In collaboration with Dr. XG Xi of the Institut Curie Finacial supports: NSFC, MOST and CAS http://softmatter.iphy.ac.cn