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Institute of Physics Chinese Academy of Sciences Beijing, China

A single molecule study on the mechanism of UvrD helicase. Ming Li ( 李 明 ) mingli@iphy.ac.cn. Institute of Physics Chinese Academy of Sciences Beijing, China. People are used to thinking about biological problems in a single molecular way. From DNA, via RNA, to protein.

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Institute of Physics Chinese Academy of Sciences Beijing, China

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

  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 T4 ligase is used to connect digoxigenin

  8. Force measurement Over damped pendulum <L> F=2.0 pN Fmag F=13.0 pN x

  9. DNA follows the WLC model

  10. Twisting DNA

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

  12. and mismatch repair.

  13. Cell

  14. Nature Reviews

  15. Dimer or monomer? The mechanism?

  16. Experimental design

  17. Unwinding Rezipping Binding Expected observations handle hairpin M-bead magnet

  18. unwinding-rezipping events

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

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

  21. A different mechanism for UvrD

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

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

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

  25. Binding kinetics

  26. K1=0.23 ±0.05 /s; K2=0.38 ±0.08 /s @ [UvrD]=5 nM

  27. K1=0.05 /s; K2=0.07 /s @ [UvrD]=1 nM 1/K1=20 Sec; 1/K2=14 Sec K -1=0.12 /s @ [UvrD]=1 nM 1/K -1=8.3 Sec

  28. Two binding events at the DNA junction 3’ 5’ 3’ 5’ 3’ 5’ unwinding loading sticking dimerizing

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

  30. Details of the unwinding events

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

  32. 3’ 5’ binding loading 3’ 5’ unwinding unbinding

  33. 3’ 5’ binding loading 3’ 5’ unwinding rewinding

  34. 3’ 5’ pausing binding 3’ 5’ unwinding unbinding

  35. 3’ 5’ 3’ 5’ binding slow rewinding 3’ 5’ unwinding fast rewinding

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

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

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

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

  40. Configurational change bends the ssDNA tail by ~50deg. v=v0 exp(-F*d/kBT) v0=68 bp/s; JMB(2003) d=0.7 nm d~0.7 nm

  41. Biological significance A road cleaner!

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

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

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