Packing DNA with proteins I. Christmas Biophysics Workshop led, Slovenia, December 18.-19.2007 T. Vuletic*, F. Livolant, M. Renouard, E. Raspaud J. Rädler; LMU, Munich *permanent address. Institut za fiziku, Zagreb, Croatia
Feb 05, 2016
Packing DNA with proteins
II. Christmas Biophysics WorkshopBled, Slovenia, December 18.-19.2007
T. Vuletic*, F. Livolant, M. Renouard, E. RaspaudJ. Rädler; LMU, Munich
*permanent address.Institut za fiziku, Zagreb, Croatia
• DNA replication, transcription, protection, repair in highly packed genetic material
Motivation: condensed phasesare functional structures
Sperm headProtamines++++. . . .+++
Spermine3+
Spermidine4+
• electrostatic DNA packing: oppositely charged multivalent ions/ basic proteins
Chromosome/ Histones
Lämmli, Uni Geneve
T2
Kleinschmidt et al. (1962)
Viral capsid proteins
A different packing: RecA protein
•Central domain: ATP binding
• N-terminus binds protomers
• C-term, negative
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• 352 residues; MW = 37,842 • promotes DNA strand exchange by forming nucleoprotein filament also, cleaves SOS response repressor • Homologs in Archaea, Eukaryota• Structure Function
• intracellular coaggregation of E.Coli RecA protein and DNA Levin-Zaidman et al. PNAS’00
500 nm
• make & study in vitro RecA/DNA dense phase
Helical filamentsRecA only:selfpolymers
RecA + dsDNA (ssDNA):nucleoprotein filaments
aggregates
crystals
hexamer:
Morrical & Cox Biochem. 1985• RecA selfpolymers/filaments and aggregates are not intermediates en route to nucleoprotein filaments
pitch 95Å6.2 monomer/turn
Egel
man
et a
l. PN
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DNA within filament
DNA pitch and base pair rise fixed
DNA within RecA
Egelman et al. Science’89
DNA base pair rise 5.1Å 5.1Å 3.4Å DNA pitch 95 Å 67 Å 34 Å
Shibata et al. PNAS’98
How the models correlate with known parameters of RecA+DNA complex, pitch, stoichiometry, monomers per turn?
RecA/DNA stoichiometry
recA [M]
0 5 10 15 20
Inte
grat
ed p
rote
in g
el d
ensi
ty (
a.u.
)
0
10
20
30
40
50
complexedunidentifieduncomplexedtotal
recA [M]
0 5 10 15 20
Inte
grat
ed D
NA
gel
den
sity
(a.
u.)
0
5
10
15
20
25
30
complexedunidentifieduncomplexedtotal
16 M DNA146bprecA [M]
0 5 10 15 20
Inte
grat
ed p
rote
in g
el d
ensi
ty (
a.u.
)
0
10
20
30
40
50
complexedunidentifieduncomplexedtotal
recA [M]
0 5 10 15 20
Inte
grat
ed D
NA
gel
den
sity
(a.
u.)
0
5
10
15
20
25
30
complexedunidentifieduncomplexedtotal
16 M DNA146bp
• gel densitometry
1 : 3
DNA146 bp16M
RecA [M] 0 0.6 1.25 2.5 3.75 5 7.5 10 15 20
complexes
uncomplexed DNA
unidentified
incubation 40min@37°C in buffer: 10mM Na-maleate pH 6.1 +5% glycerol +1mM MgCl2 +50mM NaCl +0.2 mM ATPS
uncomplexed RecA
staining protein
staining DNA
• samples with varying RecA/DNA ratio
complexes
RecA/DNA stoichiometry
• 90° static light scattering: not distinguishing selfpolymers and short nucleoprotein filaments
Franklin Pugh & Cox JBC 1987
I 90°
dsDNA 5M
1 : 3
dsDNA 3.5M
1 : 3
I 90°
• samples with varying RecA/DNA ratio
146bp fragments 11 kbp plasmid
incubation 30min@37°C in buffer TrisCl 20 mM, pH7.550 mM NaCl+5%glycerol+0.2-0.4 mM ATPS+1-10 mM Mg++
RecA/DNA stoichiometry
• complexation assay: label DNA with DAPI, then bind RecA Zaitsev NAR1998
1 : 2
• fluorescence signal decreases due to DAPI being displaced from DNA
incubation 30min@37°C in buffer: 10mM Na-maleate pH 6.1 +5% glycerol +1mM MgCl2 +50mM NaCl +0.2 mM ATPS
• 1:2 stoichiometry for both long and short DNA• 1:2 at variance with expected 1:3• is there some RecA not capable of binding?
• samples with varying RecA/DNA ratio
Kinetics by FCS and fluorimetry• FLUORIMETRY – DAPI displacement assay
• FCS – binding of RecA protein to dsDNA results in halving of diffusion constant DrodDrod=kBT*[ln (L/r) – 0.30]/( 3 L)
• r 5r • L 1.5L
• nucleation rate n, directly from complexation half-time T1/2 : n=1/(T1/2 *bp)
• RecA binding to DNA: nucleation and growth process• growth phase negligible on short DNA
• much simpler than tethered molecule or AFM measurements
RecA/DNA complex: Visualization
• TEM: visualization of RecA bound to short DNA fragments
70-80 nm short rods of helical simetry in analogy to DNA a dense phase:
liquid crystal
incubation 30min@37°C in buffer: 10mM Na-maleate pH 6.1 +5% glycerol +1mM MgCl2 +50mM NaCl +0.2 mM ATPS
• DNA 146bp=50nm; with 50% extension upon RecA binding=75nm
cholesteric hexagonal orthorhombic
Concn.(mg/ml)
Interhelix distance (Å)
160 380 670 1055
49 3231,5 23,729 a=24,09b=39,33
a=20,77b=29,72
2D 3D
Liquid crystalline phases 3D crystals
isotropic
50nm DNA fragmentsDNA liquid crystals
(Livolant, Leforestier, Luzzatti, Rill, Robinson, Strzelecka …)
RecA vs. DNA liquid crystalsDS
CN28
94.jp
g
100 m
RecA ~100 g/L
cholesteric droplets in isotropic matrix (and vice versa)
DNA 146bp > 160 g/L?
P/2 ~17 m
50 m SL03#10_005.jpg
RecA ~80 g/L
P/2 ~3-6 m
DSCN
2906
.jpg
RecA vs. DNA liquid crystalsDS
CN28
94.jp
g
100 m
RecA ~100 g/L
identifying by birefringence
DNA 146bp ~ 200 g/L
P/2 ~17 m
10 m
SL28VIIIFL8DNA_012.jpg
P/2 ~2 m
insert birefringent –plate:
positive
negative
birefringence
RecA vs. DNA liquid crystal
RecA ~60 g/L
drop
128.
jpg
50 m
section planes:sample droplet surface freeze fracture plane
section planes oblique to cholesteric stratification – arched pattern
P/2 ~100 m P/2 ~3 m
• signature of cholesteric organization at 60 g/L recA
DNA ~200 g/L
Leforestier BPJ 1993
RecA+DNA liquid crystalsmall RecA+DNA germs – columnar hexagonal phase?
17#0
1_06
6.jp
g
17#0
1_06
7.jp
g
3 mM RecA+18 mM DNA (DNA in double excess )incubation 60min@37°C in buffer: 10mM Tris-Cl pH 7.2 +10% glycerol +1mM MgCl2 +50mM NaCl +2 mM ATPS
17#0
1_06
8.jp
g
100 m
plate @ 45°
RecA+DNA liquid crystal
complexation at high DNA/RecA concentrations ~100 g/L
nematic textures: cholesteric twist is prevented by anchoring/confinement effects of slide/coverslip.
SL02
#09_
000.
jpg
60 m
SL04
#30_
118.
jpg
complexation at low DNA/RecA concentrations ~6 g/L, with subsequent concentration to ~100 g/L
next steps & prospects
• a paper in preparation, presenting kinetics/nucleation rate results • quantify the conditions for formation of RecA/DNA liq. crystals• young scientist start-up project prepared, Cro-funding• Idea: use RecA assisted hybridization on DNA chips, building on newly acquired RecA know-how and implicating people from Zagreb, Orsay, Munich and Stuttgart
end