Observation of RecBCD helicase/nuclease activity

1. Observation of RecBCD helicase/nuclease activity Bianco et al., Nature (2001) 409: 374-378.

2. Problems with using flow fields: a non-linear enzyme rate? Bianco et al., Nature (2001) 409: 374-378.

3. UvrD unzips DNA without chewing it up (conversion assay) Dessinges et al., PNAS (2004) 101: 6439-6444

4. At low force DNA hybridization is a problem Dessinges et al., PNAS (2004) 101: 6439-6444

5. Unzipping, zipping and hybridization are observed tu ~ tz ~ 1s KM, ATP ~ 50 mM Vumax ~ Vzmax ~ 250 bp/s Dessinges et al., PNAS (2004) 101: 6439-6444

6. variance = D mean Measuring step-size by variance analysis Like a random walk: N steps with a probability P (small) of moving forward a distance D Repeat the walk a large number of times and average the results together mean distance travelled = NPD variance of distance travelled = NP(1-P)D2 For UvrD, D ~ 6 +/- 1.5 bp

7. Single-molecule studies of DNA compaction Phages, Nucleosomes, Chromatin, Chromsomes,

8. Condensed states of DNA • 10s of microns of DNA length are stored in virus heads only tens of nm across. • Meter lengths of DNA are stored in sperm heads and cell nuclei, tens of microns across.

9. Viral packaging of DNA (D.E. Smith et al. Nature 413 p.748, 2001)

10. Packaging in force-clamp mode F=5 pN; ~5.5 minutes to package f29 genome (~19kb) (D.E. Smith et al. Nature 413 p.748, 2001)

11. SLIPS 0% packaged Stall Force STALLS ~30% packaged (2kb or 0.5 microns) Packaging without feedback (D.E. Smith et al. Nature 413 p.748, 2001) Force increases as bead gets pulled out of trap.

12. External force vs. velocity data No feedback, low filling A ~15 pN offset is observed between low and high-filling V vs. External Force data (D.E. Smith et al. Nature 413 p.748, 2001)

13. Internal pressure counteracts packing (~ 6 MPa pressure) 2x104 kBT for packaging 20kb phage genome (similar to results From num.sim) (D.E. Smith et al. Nature 413 p.748, 2001)

14. Force-velocity behavior V ~ Vmax exp(-FD/kBT) Vmax ~ 100 bp/s D ~ 0.1 nm (D.E. Smith et al. Nature 413 p.748, 2001)

15. Pause frequency, but not duration, is force- dependent This suggests entry into a paused state is mechanosensitive, but not exiting from it. (D.E. Smith et al. Nature 413 p.748, 2001)

16. Two other DNA translocases FtsK EcoRI 124

17. FtsK helps pump bacterial DNA Saleh et al., EMBO J. (2004) 23, 2430–2439,

18. Forward translocation, dissociation, and reverse translocation Vmax ~ 7 kbp/s!! Saleh et al., EMBO J. (2004) 23, 2430–2439,

19. Ejecting histones by applied force 17 repeats of the sea urchin 5s positionning element = 17 nucleosomes at saturation Brower-Towland et al., PNAS (2001) 99 1960-1965

20. Single dissociation events detected Brower-Towland et al., PNAS (2001) 99 1960-1965

21. Ejection is partially reversible Brower-Towland et al., PNAS (2001) 99 1960-1965

22. Force-dependence of dissociation Brower-Towland et al., PNAS (2001) 99 1960-1965

23. …dissociation of what? Brower-Towland et al., PNAS (2001) 99 1960-1965

24. DNA packaging by the X. laevis Condensin complex From Bazzett-Jones et al., Mol. Cell9 1183-1190 (2002)

25. Proposed Model From Bazzett-Jones et al., Mol. Cell9 1183-1190 (2002)

26. DNA Compaction by X. laevis condensin From Strick et al., Curr. Bio. 14 874-880 (2004)

27. Direct observation of DNA compactionby mitotic condensin a) Condensin + ATP b) Condensin - ATP c) Condensin + AMPPNP d) Interphasic condensin + ATP e) Condensin + ATP + competitor DNA

28. Mean SD n 80 40 214 70 50 110 70 40 66 Assembly of condensin onto supercoiled DNA Skewed distributions with peak at ~ 60 nm

29. Disassembly of DNA-bound condensin (“yanking”) Mean SD n 85 110 185 Skewed distribution with peak at ~ 30 nm