DNA unwinding by helicases
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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.

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DNA unwinding by helicases

Maria Mañosas

Croquette-Bensimon lab

ENS France


Outline
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

  • 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


Magnetic tweezers to study helicase activity



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 DnaB-like family, active as a hexameric ring.

Dong et al, JBC 1995



Force dependence: passive helicase

the force applied on the DNA substrate assists unwinding

where

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
Helicase and polymerase coupling

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)


Recq from e coli
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

Crystal structure of E. coli RecQ catalytic core (DA Bernstein et al 2003 EMBO)

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 12655-12663.):

.Oligomeric state: monomeric and multimeric.

. 3’-5’ polarity

.Unwinding rates ranging from 2 to 80 bp/s


Different substrates

SL hairpin: 7Kb hairpin

SS hairpin : 1.2Kb hairpin

RecQ+ATP

Z

Z

Gap substrate: 11Kb dsDNA with a 27 bases gap

RecQ+ATP

Z

Z


Two regimes of unwinding
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
Complex rezipping

SS hairpin Force=9pN

[RecQ]=1nM [ATP]=0.5mM

Slow rehybridization

Pause

Unwinding

Fast rehybridization


Non productive binding
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]=0.5nM

[ATP]=0

SS hairpin

[RecQ]=0

[ATP]=0

Unfolded

Hairpin

force

force

Folded

Hairpin


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.



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


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