TVXmasWorkshop07B
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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 • DNA replication, transcription, protection, repair in highly packed genetic material Chromosome/ Histones Viral capsid proteins Sperm head Protamines++++. . . .+++ Spermine3+ T2 4+ Spermidine Kleinschmidt et al. (1962) Lämmli, Uni Geneve • electrostatic DNA packing: oppositely charged multivalent ions/ basic proteins A different packing: RecA protein • 352 residues; MW = 37,842 • promotes DNA strand exchange by forming nucleoprotein filament also, cleaves SOS response repressor • intracellular coaggregation of E.Coli RecA protein and DNA Levin-Zaidman et al. PNAS’00 • Homologs in Archaea, Eukaryota • Structure Function www.callutheran.edu •Central domain: ATP binding • N-terminus binds protomers • C-term, negative 500 nm • make & study in vitro RecA/DNA dense phase Helical filaments RecA + dsDNA (ssDNA): nucleoprotein filaments crystals aggregates pitch 95Å 6.2 monomer/turn Egelman et al. PNAS’01 RecA only: selfpolymers hexamer: • RecA selfpolymers/filaments and aggregates are not intermediates en route to nucleoprotein filaments Morrical & Cox Biochem. 1985 Shibata et al. PNAS’98 DNA within filament Egelman et al. Science’89 DNA base pair rise 5.1Å 5.1Å DNA pitch and base DNA pitch 95 Å 67 Å pair rise fixed DNA within RecA 3.4Å 34 Å How the models correlate with known parameters of RecA+DNA complex, pitch, stoichiometry, monomers per turn? RecA/DNA stoichiometry RecA [M] 0 0.6 1.25 2.5 3.75 5 7.5 10 15 20 staining protein complexes uncomplexed RecA Integrated protein gel density (a.u.) • gel densitometry 50 total uncomplexed unidentified complexed 40 30 20 10 0 complexes unidentified DNA 146 bp 16M • 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 uncomplexed DNA Integrated DNA gel density (a.u.) staining DNA 30 0 5 1:3 25 10 15 20 recA 16[M] M DNA146bp 20 15 total uncomplexed unidentified complexed 10 5 0 0 5 10 recA [M] 15 20 RecA/DNA stoichiometry 146bp fragments 11 kbp plasmid dsDNA 3.5M 1:3 I90° I90° 1:3 dsDNA 5M 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 pH6.1, DNA 3M +RecA: 30 min @37°C 4000 I345/455nm (a.u.) pool A B DNA DNA146 2000 • 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 0 0 1 2 3 4 recA [M] • 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 I345/455nm (a.u.) • FLUORIMETRY – DAPI displacement assay 4000 RecA 0.75 M + 3.0 M DNA 146bp Fluorimetry: DAPI displacement assay • RecA binding to DNA: nucleation and growth process T1/2=240 s 3000 • growth phase negligible on short DNA D (s) 37°C pH6.15 1000 T1/2=120-300 s Fluorescence correlation spectroscopy RecA 1.2 M + 1.5 M DNA 210bp 0 0 10 20 30 incubation time [min] 40 • FCS – binding of RecA protein to dsDNA results in halving of diffusion constant Drod Drod=kBT*[ln (L/r) – 0.30]/( 3 p h L) • r 5r • L 1.5L • nucleation rate n, directly from complexation half-time T1/2 : n=1/(T1/2 *bp) • much simpler than tethered molecule or AFM measurements RecA/DNA complex: Visualization • TEM: visualization of RecA bound to short DNA fragments • DNA 146bp=50nm; with 50% extension upon RecA binding=75nm incubation 30min@37°C in buffer: 10mM Na-maleate pH 6.1 +5% glycerol +1mM MgCl2 +50mM NaCl +0.2 mM ATPgS short rods of helical simetry in analogy to DNA a dense phase: liquid crystal DNA liquid crystals Concn. (mg/ml) 380 160 50nm DNA fragments 2D 3D 670 1055 isotropic Interhelix distance (Å) 49 32 31,5 cholesteric Liquid crystalline phases (Livolant, Leforestier, Luzzatti, Rill, Robinson, Strzelecka …) 29 hexagonal 23,7 a=24,09 b=39,33 a=20,77 b=29,72 orthorhombic 3D crystals RecA vs. DNA liquid crystals cholesteric droplets in isotropic matrix (and vice versa) RecA ~80 g/L RecA ~100 g/L P/2 ~17 m DSCN2906.jpg DSCN2894.jpg 100 m DNA 146bp > 160 g/L? P/2 ~3-6 m 50 m SL03#10_005.jpg RecA vs. DNA liquid crystals identifying by birefringence RecA ~100 g/L insert birefringent –plate: DSCN2894.jpg 100 m positive P/2 ~17 m birefringence DNA 146bp ~ 200 g/L P/2 ~2 m 10 m negative SL28VIIIFL8DNA_012.jpg RecA vs. DNA liquid crystal 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 50 m RecA ~60 g/L P/2 ~100 m P/2 ~3 m DNA ~200 g/L Leforestier BPJ 1993 RecA+DNA liquid crystal small RecA+DNA germs – columnar hexagonal phase? 17#01_068.jpg 100 m 17#01_066.jpg 17#01_067.jpg plate @ 45° 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 ATPgS RecA+DNA liquid crystal nematic textures: cholesteric twist is prevented by anchoring/confinement effects of slide/coverslip. complexation at high DNA/RecA concentrations ~100 g/L SL02#09_000.jpg SL04#30_118.jpg 60 m 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
