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

2. Outline IntroductionIntroduction Results on Gp41 replicative helicaseResults on Gp41 replicative helicase Results on RecQ helicaseResults 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. (A)Stepping Mechanism: two different sites of DNA binding (inchworm and rolling) (B) Brownian motor mechanism: only one binding side Unidirectional translocation

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

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 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 coupling

16. RecQ from E.Coli Family of RecQ helicases are conserved from bacteria to human.Family of RecQ helicases are conserved from bacteria to human. Essential for the maintenance of DNA integrity, playing a role in DNA repair and recombinationEssential 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 ( ): 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..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

17. Different substrates

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

20. Non-productive binding SS hairpin [RecQ]=0 [ATP]=0 SS hairpin [RecQ]=0.5nM [ATP]=0 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.

21. Measuring the binding constant and the cooperative factor from ssDNA elasticity measurements. Measuring the binding constant and the cooperative factor from ssDNA elasticity measurements. [ATP]=0.5mM K d =0.44±0.05nM n=1.7±0.1 Θ=L [RecQ] -L 0 /L ∞ -L 0 =[RecQ] n /([RecQ] n +K d ) Force=1pN Binding properties

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 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. 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: Regime 1

24. Measuring translocation rate V trans =N b /T=80±8b/s SS hairpin [RecQ]=0.1nM [ATP]=0.5mM Translocation along ssDNA Molecular extension Force NbNb T 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. Unwinding Pauses Switch Regime 2: complex 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 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. RecQ Gp41 RecQ Gp41

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