DNA unwinding by helicases Maria Mañosas Croquette-Bensimon lab ENS France

1. DNA unwinding by helicases Maria Mañosas Croquette-Bensimon lab ENS France

2. Outline • Introduction • Results on Gp41 replicative helicase • Results on RecQ helicase

3. Importance of DNA unwinding DNA replication Transcription DNA repair and recombination

4. 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

5. 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

6. 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.

7. Models for helicase activity Unwinding: passive versus active Betterton and Jülicher Phys. Rev. E, 2005 Unwinding rate of passive helicase

8. Magnetic tweezers to study helicase activity

9. Unzipping DNA

10. 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

11. Gp41 replicative helicase gp41 helicase: 5’-3’ polarity, belongs to DnaB-like family, active as a hexameric ring. Dong et al, JBC 1995

12. Tracking Unwinding and translocation activities

13. Force dependence: passive helicase the force applied on the DNA substrate assists unwinding where T. Lionnet et al PNAS 2007

14. Sequence dependence Using the rate dependence to sequencing DNA K. Herbert et al Cell 2006

15. 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)

16. 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

17. 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

18. 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

19. Complex rezipping SS hairpin Force=9pN [RecQ]=1nM [ATP]=0.5mM Slow rehybridization Pause Unwinding Fast rehybridization

20. 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

21. 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

22. 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

23. 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

24. 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.

25. Sequence dependence

26. Pauses pause ZOOM

27. Regime 2: complex unwinding Pauses Switch Unwinding

28. 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.

29. Acknowledges Ecole Normale Superieure Timothée Lionnet Vincent Croquette David Bensimon Pennsylvania State University Michelle Spiering Steve Benkovic Funding