Institute of Physics Chinese Academy of Sciences Beijing, China Ming Li ( 李 明 ) A single molecule study on the mechanism of UvrD helicase

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

Magnetic tweezers

Genes are duplicated before cell division

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

The machine To CCD

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

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

DNA follows the WLC model

Twisting DNA

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

and mismatch repair.

Cell

Nature Reviews

Dimer or monomer? The mechanism?

Experimental design

Binding UnwindingRezipping Expected observations handle hairpin M-bead magnet

unwinding-rezipping events

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

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

A different mechanism for UvrD

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

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

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

Binding kinetics

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

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

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

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

Details of the unwinding events

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

3’ 5’ 3’ 5’ loading binding unwinding unbinding

3’ 5’ 3’ 5’ loading binding unwinding rewinding

3’ 5’ 3’ 5’ binding unwinding pausing unbinding

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

4) Dimer undergoes a conformational change to become active.

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

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

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

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.

Biological significance A road cleaner!

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

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

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

Acknowledgement Thank you! In collaboration with Dr. XG Xi of the Institut Curie Finacial supports: NSFC, MOST and CAS