Observation of RecBCD helicase/nuclease activity

Observation of RecBCD helicase/nuclease activity

Bianco et al., Nature (2001) 409: 374-378.

Problems with using flow fields: a non-linear enzyme rate?

Bianco et al., Nature (2001) 409: 374-378.

UvrD unzips DNA without chewing it up

(conversion assay)

Dessinges et al., PNAS (2004) 101: 6439-6444

At low force DNA hybridization is a problem

Dessinges et al., PNAS (2004) 101: 6439-6444

Unzipping, zipping and hybridization are observed

Vumax ~ Vzmax ~ 250 bp/s KM, ATP ~ 50 M u ~ z ~ 1s

Dessinges et al., PNAS (2004) 101: 6439-6444

Measuring step-size by variance analysis

mean distance travelled = NPvariance 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

meanvariance

=

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 invirus 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 package29 genome (~19kb)

(D.E. Smith et al. Nature 413 p.748, 2001)

StallForce STALLS

Packaging without feedback

Force increasesas bead gets pulledout 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

2x104 kBT forpackaging 20kbphage genome(similar to resultsFrom num.sim)

(~ 6 MPa pressure)

(D.E. Smith et al. Nature 413 p.748, 2001)

Force-velocity behavior

Vmax ~ 100 bp/s

~ 0.1 nm

V ~ Vmax exp(-F/kBT)

(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

FtsKEcoRI 124

FtsK helps pump bacterial DNA

Saleh et al., EMBO J. (2004) 23, 2430–2439,

Forward translocation, dissociation, and reverse translocation

Vmax ~ 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) 99 1960-1965

Single dissociation events detected

Brower-Towland et al., PNAS (2001) 99 1960-1965

Ejection is partially reversible

Brower-Towland et al., PNAS (2001) 99 1960-1965

Force-dependence of dissociation

Brower-Towland et al., PNAS (2001) 99 1960-1965

…dissociation of what?

Brower-Towland et al., PNAS (2001) 99 1960-1965

DNA packaging by the X. laevis Condensin complex

From Bazzett-Jones et al., Mol. Cell 9 1183-1190 (2002)

Proposed Model

From Bazzett-Jones et al., Mol. Cell 9 1183-1190 (2002)

DNA Compaction by X. laevis condensin

From Strick et al., Curr. Bio. 14 874-880 (2004)

Direct observation of DNA compaction

by mitotic condensina) 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

80 40 214

70 50 110

70 40 66

Skewed distributions

with peak at ~ 60 nm

Disassembly of DNA-bound condensin (“yanking”)

85 110 185

Mean SD n

Skewed distribution

with peak at ~ 30 nm