Packing DNA with proteins

II. Christmas Biophysics WorkshopBled, Slovenia, December 18.-19.2007

T. Vuletic*, F. Livolant, M. Renouard, E. Raspaud

J. 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

ww

w.c

allu

thera

n.e

du

• 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

Ege

lman

et a

l. P

NA

S’0

1

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

complexedunidentifieduncomplexed

total

recA [M]

0 5 10 15 20

Inte

grat

ed D

NA

gel

den

sity

(a.

u.)

0

5

10

15

20

25

30

complexedunidentified

uncomplexed

total

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

complexedunidentifieduncomplexed

total

recA [M]

0 5 10 15 20

Inte

grat

ed D

NA

gel

den

sity

(a.

u.)

0

5

10

15

20

25

30

complexedunidentified

uncomplexed

total

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 23,729a=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 crystalsD

SC

N2

89

4.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

DS

CN

29

06

.jp

g

RecA vs. DNA liquid crystalsD

SC

N2

89

4.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

dro

p1

28

.jp

g

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 crystal

small RecA+DNA germs – columnar hexagonal phase?

17

#0

1_0

66

.jp

g

17

#0

1_0

67

.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_0

68

.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.

SL0

2#

09

_00

0.jp

g

60 m

SL0

4#

30

_11

8.jp

g

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