observation of recbcd helicase/nuclease activity bianco et al., nature (2001) 409: 374-378
TRANSCRIPT
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
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 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