Nuclear Uptake of DNA
H Salman, D Zbaida, D Chatenay, Y Rabin, M Elbaum. PNAS 98: 7247 (2001).
Central dogma:
DNA
RNA
Nucleus
Protein
Cytoplasm
Central dogma:
DNA
RNA
Nucleus
... and beyond:
Protein
Cytoplasm
RNA
DNA
Nuclear import of DNA:
animal virus use the host cell nucleus
DNA virus, single-strand, double-strand
RNA virus, with reverse transcription
short genome: circular plasmid, intact virion entry
long genome: linear plasmid, virion disassembly
genetic “therapy”
insertion of exogenous genes
hereditary diseases
cancer
allergies
genetic modification of plants, lab & agriculture
Agrobacterium tumefaciens
Nuclear import of DNA:
DNA 6800 b.p. plasmid
Golan et.al., Biochemistry
1999, 38, 14069.
Condensed DNA toroid
Golan et.al., ibid.
Compare protein and DNA import to cell-free nuclei:
DNA Entry through NPC
ssDNA
three possibilities
dsDNA
NLS
C
N
C
N
F
Does DNA import involve/require:
NLS ??
NLS-associated transport receptors?
an ATPase motor?
condensation or chromatinization?
What are the kinetics, mechanical forces?
Optical tweezers:
CCD
Laser Source - 830nm
A. Ashkin, Methods Cell Biol 1998, 55: 1-27.
Single molecule manipulation of DNA
anti-digoxygenin bead
U
T: U-dig
U
U
λ phage DNA, 17 micron length
U
U
T U-biotin
streptavidin, 70 kD
U
U
NLS
NLS
NLS
DNA stability in Xenopus egg extract
DNA uptake kinetics
Polymer translocation through a pore
configurational entropy: force imbalance toward longer side.
but DNA uptake slows down toward the end: effect of the bead.
force scales: Brownian force on bead
polymer deformation
F ~ k B T /3  m  ≈ 1fN
F ~ k B T / L p ≈ 80 fN
DNA uptake force?
Hold bead at partial extension. Use DNA as “calibrated spring”.
Stretching a Polymer
10
6
R2
1
2
1100 fN
4
a
x
LM
( µ m)
8
160 fN
2
x
50 fN
0
0
600
1200
t (sec)
1800
2400
L
k BT 1
x −2 1 x
F  L F(=L) = k T [[ 1 (1−
1−
1 +−x ]  ]
x
)
−
LLp 44 L L 4 L 4 L
−2
B
p
measurable (but slow!) progress for
F = 50 fN
upper limit to uptake force.
scale:
kB T
Lp
= 80 fN
Simple kinetic model
stretch
φ
∆µ
Conclusions (of the time):
The NPC is not a barrier to uptake of DNA.
fluorescence images
Nuclear uptake of DNA is distinct from NLS-mediated protein import.
does not require ATP: no ATPase motor protein
not assisted by “classical” NLS-bearing proteins
Evidence for serpentine passage though the pore.
continuous, monotonic uptake of linear dsDNA
Measured kinetics suggest a passive mechanism of uptake.
slow compared to hydrodynamic scales, stall forces < 50 fN
attached bead yields clues to dynamics
see Z Farkas, I Derenyi, T Vicsek. J. Phys. Cond. Mat. 15 (2003) S1767.