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

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

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

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

Unzipping, zipping and hybridization are observed V umax ~ V zmax ~ 250 bp/s K M, ATP ~ 50  M  u ~  z ~ 1s Dessinges et al., PNAS (2004) 101:

Measuring step-size by variance analysis mean distance travelled = NP  variance of distance travelled = NP(1-P)  2 Like a random walk: N steps with a probability P (small) of moving forward a distance  Repeat the walk a large number of times and average the results together mean variance =  For UvrD,  ~ 6 +/- 1.5 bp

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

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.

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

Packaging in force-clamp mode F=5 pN; ~5.5 minutes to package  29 genome (~19kb) (D.E. Smith et al. Nature 413 p.748, 2001)

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

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)

Internal pressure counteracts packing 2x10 4 k B T for packaging 20kb phage genome (similar to results From num.sim) (~ 6 MPa pressure) (D.E. Smith et al. Nature 413 p.748, 2001)

Force-velocity behavior V max ~ 100 bp/s  ~ 0.1 nm V ~ V max exp(-F  /k B T) (D.E. Smith et al. Nature 413 p.748, 2001)

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)

Two other DNA translocases FtsK EcoRI 124

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

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

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

Single dissociation events detected Brower-Towland et al., PNAS (2001)

Ejection is partially reversible Brower-Towland et al., PNAS (2001)

Force-dependence of dissociation Brower-Towland et al., PNAS (2001)

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

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

Proposed Model From Bazzett-Jones et al., Mol. Cell (2002)

DNA Compaction by X. laevis condensin From Strick et al., Curr. Bio (2004)

Direct observation of DNA compaction by mitotic condensin a) Condensin + ATP b) Condensin - ATP c) Condensin + AMPPNP d) Interphasic condensin + ATP e) Condensin + ATP + competitor DNA

Assembly of condensin onto supercoiled DNA Mean SD n Skewed distributions with peak at ~ 60 nm

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