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