Packing DNA with proteins

II. Christmas Biophysics Workshop Bled, Slovenia, December 18.-19.2007 Packing DNA with proteins T.Vuletic*, F. Livolant, M. Renouard, E. Raspaud J. Rädler; LMU, Munich *permanent address. Institut za fiziku, Zagreb, Croatia

Motivation: condensed phases are functional structures Protamines++++. . . .+++ Spermine3+ Spermidine4+ Chromosome/ Histones Sperm head Viral capsid proteins Kleinschmidt et al. (1962) Lämmli, Uni Geneve • DNA replication, transcription, protection, repair in highly packed genetic material T2 • electrostatic DNA packing: • oppositely charged multivalent ions/ basic proteins

A different packing: RecA protein • intracellular coaggregation of E.Coli RecA protein and DNA • 352 residues; MW = 37,842 • promotes DNA strand exchange • by forming nucleoprotein filament also, cleaves SOS response repressor • • Homologs in Archaea, Eukaryota • Structure  Function Levin-Zaidman et al. PNAS’00 • Central domain: ATP binding www.callutheran.edu • make & study in vitro RecA/DNA dense phase 500 nm • C-term, negative • N-terminus binds protomers

Helical filaments hexamer: RecA + dsDNA (ssDNA): nucleoprotein filaments RecA only: selfpolymers aggregates crystals pitch 95Å 6.2 monomer/turn Egelman et al. PNAS’01 • RecA selfpolymers/filaments and aggregates are not intermediates en route to nucleoprotein filaments Morrical & Cox Biochem. 1985

DNA within filament Shibata et al. PNAS’98 Egelman et al. Science’89 DNA base pair rise 5.1Å 5.1Å 3.4Å DNA pitch 95 Å 67Å 34 Å DNA pitch and base pair rise fixed How the models correlate with known parameters of RecA+DNA complex, pitch, stoichiometry, monomers per turn? DNA within RecA

RecA/DNA stoichiometry • gel densitometry 16 mM DNA146bp 16 mM DNA146bp 1 : 3 RecA [mM] 0 0.6 1.25 2.5 3.75 5 7.5 10 15 20 complexes staining protein uncomplexed RecA staining DNA complexes unidentified DNA 146 bp 16mM uncomplexed DNA • samples with varying RecA/DNA ratio incubation 40min@37°C in buffer: 10mM Na-maleate pH 6.1 +5% glycerol +1mM MgCl2 +50mM NaCl +0.2 mM ATPgS

RecA/DNA stoichiometry 146bp fragments 11 kbp plasmid dsDNA 3.5mM 1 : 3 1 : 3 I90° I90° dsDNA 5mM Franklin Pugh & Cox JBC 1987 • 90° static light scattering: not distinguishing selfpolymers and short nucleoprotein filaments • samples with varying RecA/DNA ratio incubation 30min@37°C in buffer TrisCl 20 mM, pH7.5 50 mM NaCl+5%glycerol+0.2-0.4 mM ATPgS+1-10 mM Mg++

RecA/DNA stoichiometry • complexation assay: label DNA with DAPI, then bind RecA Zaitsev NAR1998 • fluorescence signal decreases due to DAPI being displaced from DNA • 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? 1 : 2 • samples with varying RecA/DNA ratio incubation 30min@37°C in buffer: 10mM Na-maleate pH 6.1 +5% glycerol +1mM MgCl2 +50mM NaCl +0.2 mM ATPgS

Kinetics by FCS and fluorimetry • FLUORIMETRY – DAPI displacement assay • RecA binding to DNA: nucleation and growth process • growth phase negligible on short DNA • nucleation rate n, directly from complexation half-time T1/2 : n=1/(T1/2 *bp) • much simpler than tethered molecule or AFM measurements • FCS – binding of RecA protein to dsDNA results in halving of diffusion constant Drod Drod=kBT*[ln (L/r) – 0.30]/( 3p hL) • r 5r • L  1.5L

RecA/DNA complex: Visualization • TEM: visualization of RecA bound to short DNA fragments • DNA 146bp=50nm; with 50% extension upon RecA binding=75nm 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 ATPgS

DNA liquid crystals 50nm DNA fragments Concn. (mg/ml) 380 160 670 1055 2D 3D isotropic a=24,09 b=39,33 a=20,77 b=29,72 Interhelix distance (Å) 49 32 31,5 29 23,7 cholesteric hexagonal orthorhombic Liquid crystalline phases 3D crystals (Livolant, Leforestier, Luzzatti, Rill, Robinson, Strzelecka …)

RecA vs. DNA liquid crystals 50 mm cholesteric droplets in isotropic matrix (and vice versa) RecA ~80 g/L RecA ~100 g/L 100 mm DSCN2906.jpg DSCN2894.jpg P/2 ~17 mm DNA 146bp > 160 g/L? P/2 ~3-6 mm SL03#10_005.jpg

RecA vs. DNA liquid crystals 10 mm identifying by birefringence RecA ~100 g/L insert birefringent l –plate: 100 mm DSCN2894.jpg positive P/2 ~17 mm birefringence DNA 146bp ~ 200 g/L negative P/2 ~2 mm SL28VIIIFL8DNA_012.jpg

RecA vs. DNA liquid crystal 50 mm section planes oblique to cholesteric stratification – arched pattern • signature of cholesteric organization at 60 g/L recA section planes: sample droplet surface freeze fracture plane drop128.jpg P/2 ~100 mm P/2 ~3 mm Leforestier BPJ 1993 RecA ~60 g/L DNA ~200 g/L

RecA+DNA liquid crystal small RecA+DNA germs – columnar hexagonal phase? 100 mm lplate @ 45° 17#01_068.jpg 17#01_067.jpg 17#01_066.jpg 3mM 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 ATPgS

RecA+DNA liquid crystal 60 mm nematic textures: cholesteric twist is prevented by anchoring/confinement effects of slide/coverslip. SL04#30_118.jpg SL02#09_000.jpg complexation at high DNA/RecA concentrations ~100 g/L 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 acquiredRecA know-how and implicating people from Zagreb, Orsay, Munich and Stuttgart

end

Packing DNA with proteins