institute of physics chinese academy of sciences beijing, china ming li ( 李 明 )...
TRANSCRIPT
Institute of PhysicsChinese Academy of Sciences
Beijing, China
Ming Li ( 李 明 )[email protected]
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 separatedin order for the genes to be duplicated.
The machine
To CCD
F B
Connecting DNA to a surface and a handle
Biotin ended
digoxigenin
T4 ligase is used to connect
2
Bmag
k T LF
x
Force measurement
F=2.0 pN F=13.0 pN
<L>
Fmagx
Over damped pendulum
20 0
1 1
4(1 / ) 4BK T L
FA L L L
0 02
0 0
ˆ( ) ˆ ˆ( )2
L L
BWLC
Ak T t sE ds F t s zds
s
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 mism
atch repair.
Cell
Nature Reviews
Dimer or monomer?
The mechanism?
Experimental design
Binding Unwinding Rezipping
Expected observations
handle
hairpin
M-bead
magnet
unwinding-rezipping events
Unwinding rate versus force
F=5 pNF=9 pN
F=5 pNF=9 pN
Force hinders UvrD, rather than helps it.
A force higher than ~14 pNunzips 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
1 22,k kE DNA E DNA E E DNA E DNA
1 21 2
2 1
( ) ( )k t k tk kf t e e
k k
1 21 2
2 1
( ) ( )k t k tk kf t e e
k k
K1=0.23 ±0.05 /s; K2=0.38 ±0.08 /s @ [UvrD]=5 nM
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
Two binding events at the DNA junction
3’
5’
3’
5’
3’
5’
loading unwindingsticking dimerizing
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
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=v0 exp(-F*d/kBT)v0=68 bp/s; JMB(2003)
d=0.7 nm
Configurational change bends the ssDNA tail by ~50deg.
Biologicalsignificance
Aroadcleaner!
!
Autoinhibitory 2B domain must be released to activate the helicase.
Summary
EMBO Journal 2008, 27, 3279Sun 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 CurieFinacial supports: NSFC, MOST and CAS
http://softmatter.iphy.ac.cn
