Helicases as Molecular Motors BIOC/MMG 352 Scott Morrical Dept. of Biochemistry.
DNA unwinding by helicases Maria Mañosas Croquette-Bensimon lab ENS France
description
Transcript of DNA unwinding by helicases Maria Mañosas Croquette-Bensimon lab ENS France
DNA unwinding by DNA unwinding by helicaseshelicases
Maria Mañosas Maria Mañosas Croquette-Bensimon labCroquette-Bensimon lab
ENS FranceENS France
OutlineOutline
• Introduction Introduction
•Results on Gp41 replicative Results on Gp41 replicative helicasehelicase
•Results on RecQ helicaseResults on RecQ helicase
Importance of DNA Importance of DNA unwindingunwinding
DNA replication DNA replication
TranscriptionTranscription
DNA repair and recombinationDNA repair and recombination
HelicasesHelicasesEnzymes that use the energy of ATP hydrolysis to Enzymes that use the energy of ATP hydrolysis to move unidirectionally along ssDNA and unwind move unidirectionally along ssDNA and unwind dsDNA. They play a role in every aspect of DNA dsDNA. They play a role in every aspect of DNA (RNA) metabolism (e.g replication, repair, (RNA) metabolism (e.g replication, repair, recombination….)recombination….)
..Sequence and structure (families)Sequence and structure (families)
.Oligomeric state.Oligomeric state
.Directionallity.Directionallity
.Step size.Step size
.Processivity and unwinding rate.Processivity and unwinding rate
.Passive versus active.Passive versus active
Models for helicase Models for helicase activity activity General ingredient: General ingredient: Different enzymes Different enzymes
conformation with different DNA affinities conformation with different DNA affinities associated to different ATP ligation states.associated to different ATP ligation states.
(A)(A) Stepping Stepping Mechanism: Mechanism: two two different sites of DNA different sites of DNA binding (inchworm and binding (inchworm and rolling)rolling)
(B) Brownian motor (B) Brownian motor mechanism: mechanism: only one only one binding sidebinding side
Unidirectional translocationUnidirectional translocation
Models for helicase Models for helicase activity activity Unwinding: passive versus Unwinding: passive versus
activeactive
Passive:Passive: helicase behaves helicase behaves opportunistically, relying on opportunistically, relying on the fraying of the DNA fork the fraying of the DNA fork
Active:Active: direct direct destabilization of the DNA destabilization of the DNA fork.fork.
Models for helicase Models for helicase activity activity Unwinding: passive versus Unwinding: passive versus
activeactive Betterton and Jülicher Betterton and Jülicher Phys. Rev.Phys. Rev. E, 2005 E, 2005
7/)/( 20
kekekkv G
u Unwinding rate of passive helicaseUnwinding rate of passive helicase
Magnetic tweezers Magnetic tweezers to study helicase to study helicase
activityactivity
Unzipping DNAUnzipping DNA
What’s new?What’s new?
Single molecule experiments:Single molecule experiments: measuring distributions of instead of measuring distributions of instead of measuring average propertiesmeasuring average properties
Helicase activity assisted by force: Helicase activity assisted by force: discriminating between passive and active discriminating between passive and active mechanismsmechanisms
gp41 helicase: 5’-3’ polarity, belongs to DnaB-like family, active as a hexameric ring.
Dong et al, JBC 1995
Gp41 replicative Gp41 replicative helicasehelicase
Tracking Unwinding and Tracking Unwinding and translocation activitiestranslocation activities
Force dependence: passive Force dependence: passive helicasehelicase
the force applied on the DNA substrate assists unwinding
xdffdxfxfGbp )(
)(
))(/)(())(( 0 fGGn
nu
bpek
ffkv
where
T. Lionnet et al PNAS 2007
Sequence dependenceSequence dependence
Using the rate dependence to sequencing DNAK. Herbert et al Cell 2006
Helicase and polymerase coupled activity
Holoenzyme strand displacement activity does strongly depends on the force (as helicase does)
Synthesis rates are independent of the applied force and agrees with that of the replisome measured in bulk assays (300bp/s)
Helicase and polymerase Helicase and polymerase couplingcoupling
RecQ from RecQ from E.ColiE.Coli• Family of RecQ helicases are conserved from bacteria to Family of RecQ helicases are conserved from bacteria to
human. human. • Essential for the maintenance of DNA integrity, playing a Essential for the maintenance of DNA integrity, playing a
role in DNA repair and recombinationrole in DNA repair and recombination•
Crystal structure of E. coli RecQ catalytic core (DA Bernstein et al 2003 EMBO)
Previous studies on RecQ from Previous studies on RecQ from E. ColiE. Coli ( (F.G. Harmon S.C. Kowalczykowski, J. Biol.Chem. 2001, XD Zhang et al. J. Biol. Chem. 2006 Vol 281 12655-12663.):):
.Oligomeric state: monomeric and multimeric..Oligomeric state: monomeric and multimeric. . 3’-5’ polarity. 3’-5’ polarity .Unwinding rates ranging from 2 to 80 bp/s.Unwinding rates ranging from 2 to 80 bp/s
ZZ
RecQ+ATP
RecQ+ATPZ Z
SL hairpin: 7Kb hairpin SS hairpin : 1.2Kb hairpin
Gap substrate: 11Kb dsDNA with a 27 bases gap
Different substratesDifferent substrates
Two regimes of Two regimes of unwindingunwinding
(1) Fast and processive(1) Fast and processive(2) Slow and with pausing(2) Slow and with pausing
SL hairpin Force=6pN[RecQ]=0.05nM [ATP]=0.5mM
Slow unwinding
Fast unwinding
Complex rezippingComplex rezipping
Pause
Unwinding
Slow rehybridization
Fast rehybridization
SS hairpin Force=9pN[RecQ]=1nM [ATP]=0.5mM
Non-productive binding Non-productive binding
SS hairpin[RecQ]=0 [ATP]=0
SS hairpin[RecQ]=0.5nM [ATP]=0
force force
Folded Hairpin
Unfolded
Hairpin
Experimental protocol: (i) increase the force to mechanically denaturate the hairpin(ii) decrease the force to allow the hairpin to refold.
Measuring the binding constant and the cooperative factor from Measuring the binding constant and the cooperative factor from
ssDNA elasticity measurementsssDNA elasticity measurements..
[ATP]=0.5mM Kd=0.44±0.05nM n=1.7±0.1
Θ=L[RecQ]-L0/L∞-L0=[RecQ]n/([RecQ]n+Kd)
Force=1pN
Binding properties Binding properties
Evidence for different Evidence for different oligomeric states oligomeric states
Ratio between fast regime (1) and slow regime (2) Ratio between fast regime (1) and slow regime (2) depends on [RecQ]. Regime 1 might be the activity of an depends on [RecQ]. Regime 1 might be the activity of an
oligomeric stateoligomeric state SL hairpin Force=6pN[RecQ]=0.05nM [ATP]=0.5mM
Regime 2
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
Force dependence of Unwinding: Force dependence of Unwinding: Regime 1 Regime 1
Measuring translocation rate Measuring translocation rate
Vtrans=Nb/T=80±8b/s
SS hairpin [RecQ]=0.1nM [ATP]=0.5mM
Translocation along ssDNA
Molecular extensionForce Nb
T
The translocation velocity is close to the measured unwinding velocity. RecQ is a very efficient helicase: unwinds DNA at its maximum rate.
Sequence dependence Sequence dependence
PausesPauses
pause
ZOOM
Unwinding
Pauses
Switch
Regime 2: complex unwinding Regime 2: complex unwinding
Conclusions: 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
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.
RecQGp41
RecQGp41
Acknowledges Acknowledges
Ecole Normale SuperieureTimothée LionnetVincent CroquetteDavid Bensimon
Pennsylvania State UniversityMichelle SpieringSteve Benkovic
Funding