1. Thermodynamic and kinetic characterization of proteins that destabilize duplex DNA by single molecule DNA stretching Prof. Mark C. Williams laboratory : Kiran Pant Margareta Cruceanu Leila Shortki Dana Vladescu Department of Physics Northeastern University Boston, MA, USA. Mechanics of Life workshop, June 12, 2007 Ioulia Rouzina University of Minnesota

2. Stretching single DNA molecules Microscope objectives Laser beam Glass Micropipette DNA molecule Polystyrene bead Laser beam F Torsionally relaxed DNA

3. DNA overstretching is a force-induced melting transition Equilibrium melting transition DNA melting free energy  G is area under transition region between dsDNA and ssDNA when transition is reversible ssDNA dsDNA theoretical  dsDNA stretch GG Nonequilibrium strand separation

4. HIV-1 NC destabilizes ds DNA and facilitates DNA strand recombination HIV NC No protein Extension per base pair, nm Force, pN “Fast” duplex destabilizers lower melting force and have little hysteresis

5. Poly Lysine promotes DNA strand annealing more efficiently than wt HIV-1 NC or zinc-less NC, but does not facilitate conversion DNA stretching: Increased melting force suggests lack of duplex destabilization; Very small hysteresis upon relaxation suggests strongly facilitated strand re-annealing

6. 0.2 0.3 0.4 0.5 0.6 0.7 DNA extension per bp, nm DNA stretching force, pN HTLV-1NC No protein HTLV-1 NC exhibits stretching profile characteristic of a slow single-stranded binding protein Dependence of melting force on pulling rate suggests slow protein binding to ss DNA The observed large hysteresis indicates that HTLV-1 NC does not easily dissociate single-stranded DNA Cooperativity of binding to DNA may result in slow dissociation + - + - + - + - + - negatively charged nucleic acid protein gp32 gp32* No protein

7. SSB proteins are essential for DNA replication Single-stranded DNA binding (SSB) proteins bind to ssDNA and prevent the formation of hairpin structure during DNA replication. dsDNA T4 Gene 32 http://www.molbio.uoregon.edu/~pvh/images/DNARfork.gif Bacteriophage T4 replication fork Fork movement

8. T4 Gene 32 protein (gp32) Full length gp32 Binds preferentially to ssDNA Highly cooperative binding Does not lower T m of dsDNA Fragment *I Lacks 48 C-terminal residues Lowers T m by ~ 50 °C Fragment *III Lacks C- and N-terminal domains Lacks cooperative binding Lowers T m 211 253 301 *I*I *III N -N - - C Cooperative binding to ssDNA: protein-protein interactions require N-terminus

9. *I lowers DNA melting force in high salt, gp32 does not No protein stretch gp32 stretch *I stretch gp32 relax *I relax 200 nM protein 100 mM Na + *I retains cooperative binding, lacks C-terminal domain No protein relax

10. Measurement of the equilibrium DNA melting force and relaxation kinetics kinetics in the presence of gp32 and *I Relaxation time constant Equilibrium melting force Force relaxation at constant DNA extension beyound B-DNA length

11. Single molecule measurement of equilibrium binding of gp32 and *I to ssDNA 50 mM Na + 75 mM Na + 100 mM Na + 150 mM Na + 200 mM Na + *I gp32

12. Single molecule K ss results for gp32 and *I agree with and extend previous bulk studies

13. no protein 2. Kinetics of individual gp32 binding events Melting transition occurs when: Extension rate = rate of DNA melting due to protein binding v Pulling rate k gp32 binding rate xx Increase in DNA length per base pair due to protein binding n gp32 binding site size (bp) 250 nm/s 25 nm/s 200 nM *I gp32 or *I melting

14. Kinetics of reaction from pulling rate dependence of DNA overstretching force Binding rate is product of probability of melting n base pairs ssDNA-protein association rate k a Apply force: linear approximation measured

15. n=7 ± 1 bp binding site size for gp32 from slope of F k vs ln( v ) only DNA *III 200 nM gp32 400 nM gp32 50 nM *I 100 nM *I 200 nM *I Mechanical measurement of binding site size and protein association rates k a from intercept of F k vs ln( v ) with only DNA stretching curve

16. 3 order of magnitude difference in rates between *I and gp32 Rates for *I greater than diffusion limit Stronger than linear dependence of rates on protein concentration Protein concentration dependence of k a Protein concentration (nM) Protein binding rate k a (s -1 ) 10 3 10 5 10 6 10 2 10 4 10 1 3-D diffusion limit  *I  gp32 100 mM Na +

17. Pure 1-D diffusion along DNA What happens if ? Protein finds site before dissociation Pure 1-D diffusion

18. Concentration dependence of k a K ds – binding constant to dsDNA C - protein concentration n - binding site size in bp k s – 1D sliding rate, ~10 6 s -1 K ds is obtained directly from fits to data McGhee and Von Hippel isotherm 50 mM Na + 75 mM Na + 100 mM Na + 150 mM Na + 200 mM Na + Full length gp32 Fragment *I

19. Single molecule measurement of equilibrium binding of gp32 and *I to dsDNA gp32 shows no salt dependence *I shows weaker salt dependence than at higher salt Never previously measured for *I

20. Model for salt-dependent gp32 binding to DNA gp32 binding to ssDNA is regulated by the strongly salt dependent opening of its C terminal domain (CTD) P op - probability of opening CTD, - free energy of CTD binding to DNA binding site In high Na + Unbound *I Cationic DNA binding site Core N Unbound gp32 Core N C-terminal domain (CTD) gp32 bound cooperatively to ssDNA N N DNA Core Na +

21. Another SSB case study: gp2.5 from T7 bacteriophage gp2.5 is a faster SSB then gp32 (less hysteresis; complete re-annealing)

22. CTD truncation of gp2.5 is faster then wt gp2.5 Equilibrium is reached at ~5nm/s: we can determine protein dissociation rate

23. Determining Protein Dissociation rate from rate dependence of DNA unwinding F Ln(pulling rate) k a2 k a1 kdkd F m1 F m2 C 1 <C 2 F m0

24. Alternative approach to determine protein dissociation rate by DNA relaxation Results: dissociation becomes slower in lower salt Dissociation is faster for  CTD mutant of gp2.5 Wt gp2.5  CTD gp2.5

25. Conclusions Equilibrium force-induced melting –New method for measuring binding constant to ssDNA –Results match bulk data where available, extend data to new conditions Rate dependence of overstretching force –New method for measuring protein binding rates –Explains inability of gp32 to lower T m 1D diffusion-enhanced kinetics of DNA melting –Rates governed by binding strength, diffusion, and protein conformational changes –Measurements of equilibrium binding constants to dsDNA gp32 binding regulated by salt-dependent conformational change –Governed by counterion condensation of sodium on DNA and CTD –Electrostatic model explains all data on gp32 binding for first time –CTD binding by replication proteins may also regulate DNA binding Two complementary approaches to measure protein dissociation rates from ssDNA –By force relaxation to equilibrium at constant extension after fast DNA release - By determining the slow puling rate where DNA stretching curves become rate- independent