Transcript Helicases - Maintenance

DNA unwinding by helicases
Maria Mañosas
Croquette-Bensimon lab
ENS France
Outline
• Introduction
• Results on Gp41 replicative
helicase
• Results on RecQ helicase
Importance of DNA unwinding
DNA replication
Transcription
DNA repair and recombination
Helicases
Enzymes that use the energy of ATP hydrolysis to move
unidirectionally along ssDNA and unwind dsDNA. They
play a role in every aspect of DNA (RNA) metabolism
(e.g replication, repair, recombination….)
.Sequence and structure (families)
.Oligomeric state
.Directionallity
.Step size
.Processivity and unwinding rate
.Passive versus active
Models for helicase activity
General ingredient: Different enzymes conformation
with different DNA affinities associated to different
ATP ligation states.
Unidirectional translocation
(A) Stepping Mechanism:
two different sites of
DNA binding (inchworm
and rolling)
(B) Brownian motor
mechanism: only one
binding side
Models for helicase activity
Unwinding: passive versus active
Passive: helicase behaves
opportunistically, relying on
the fraying of the DNA fork
Active: direct
destabilization of the DNA
fork.
Models for helicase activity
Unwinding: passive versus active
Betterton and Jülicher Phys. Rev. E, 2005
Unwinding rate of passive helicase
vu  k  ( /  )  k  e
 G0
2
 k e  k / 7
Magnetic tweezers
to study helicase activity
Unzipping DNA
What’s new?
Single molecule experiments: measuring
distributions of instead of measuring average
properties
Helicase activity assisted by force:
discriminating between passive and active
mechanisms
Gp41 replicative helicase
gp41 helicase: 5’-3’
polarity, belongs to DnaBlike family, active as a
hexameric ring.
Dong et al, JBC 1995
Tracking Unwinding and
translocation activities
Force dependence: passive helicase
the force applied on the DNA
substrate assists unwinding
vu  k  ( ( f ) /  ( f )) n 
k  (e
 n ( G0  Gbp ( f ))
)
where
Gbp ( f )   fx   fdx    xdf
T. Lionnet et al PNAS 2007
Sequence dependence
Using the rate dependence to sequencing DNA
K. Herbert et al Cell 2006
Helicase and polymerase coupling
Holoenzyme strand displacement
activity does strongly depends
on the force (as helicase does)
Helicase and polymerase coupled activity
Synthesis rates are independent of
the applied force and agrees with
that of the replisome measured in
bulk assays (300bp/s)
RecQ from E.Coli
• Family of RecQ helicases are conserved from bacteria to
human.
• Essential for the maintenance of DNA integrity, playing a
role in DNA repair and recombination
•
Previous studies on RecQ from E. Coli
(F.G. Harmon S.C. Kowalczykowski,
J. Biol.Chem. 2001, XD Zhang et al.
J. Biol. Chem. 2006 Vol 281 1265512663.):
.Oligomeric state: monomeric and
multimeric.
. 3’-5’ polarity
.Unwinding rates ranging from 2 to 80
bp/s
Crystal structure of E. coli RecQ
catalytic core (DA Bernstein et al
2003 EMBO)
Different substrates
SL hairpin: 7Kb hairpin
SS hairpin : 1.2Kb hairpin
RecQ+ATP
Z
Z
Gap substrate: 11Kb dsDNA
with a 27 bases gap
Z
RecQ+ATP
Z
Two regimes of unwinding
(1) Fast and processive
(2) Slow and with pausing
SL hairpin Force=6pN
[RecQ]=0.05nM [ATP]=0.5mM
Slow unwinding
Fast
unwinding
Complex rezipping
SS hairpin Force=9pN
[RecQ]=1nM [ATP]=0.5mM
Slow rehybridization
Pause
Unwinding
Fast rehybridization
Non-productive binding
Experimental protocol: (i) increase the force to mechanically denaturate the hairpin
(ii) decrease the force to allow the hairpin to refold.
SS hairpin
[RecQ]=0
[ATP]=0
force
Unfolded
Hairpin
force
Folded
Hairpin
SS hairpin
[RecQ]=0.5nM
[ATP]=0
Binding properties
Measuring the binding constant and the cooperative factor from ssDNA
elasticity measurements.
Θ=L[RecQ]-L0/L∞-L0=[RecQ]n/([RecQ]n+Kd)
Force=1pN
[ATP]=0.5mM
Kd=0.44±0.05nM
n=1.7±0.1
Evidence for different oligomeric
states
Ratio between fast regime (1) and slow regime (2) depends on [RecQ].
Regime 1 might be the activity of an oligomeric state
SL hairpin Force=6pN
[RecQ]=0.05nM [ATP]=0.5mM
Regime 2
Regime 1
Force dependence of Unwinding: Regime 1
For all DNA substrates studied and all [RecQ], the measured unwinding velocity
ranges from 60-80bp/s independently of the force applied. RecQ helicase
activity is almost independent of the applied force
Measuring translocation rate
SS hairpin
[RecQ]=0.1nM [ATP]=0.5mM
Molecular extension
Force
Nb
T
Translocation
along ssDNA
Vtrans=Nb/T=80±8b/s
The translocation velocity is close to the measured
unwinding velocity. RecQ is a very efficient helicase:
unwinds DNA at its maximum rate.
Sequence dependence
Pauses
pause
ZOOM
Regime 2: complex unwinding
Pauses
Switch
Unwinding
Conclusions:
Gp41 versus RecQ:
Gp41 shows as unwinding rate that critically depend on both force and
sequence. Its behaviour is well explained by a passive model
RecQ unwinding behavior (regime 1) is almost independent on the
sequence and it unwinds DNA as quick as it translocates along ssDNA
RecQ
Gp41
RecQ
Gp41
Two modes of unwinding in RecQ:
RecQ also shows another mode of unwinding ( Regime 2), which is much
slower and displays long pauses and switches. It probably corresponds to
a low oligomeric state of the protein.
Acknowledges
Ecole Normale Superieure
Timothée Lionnet
Vincent Croquette
David Bensimon
Pennsylvania State University
Michelle Spiering
Steve Benkovic
Funding