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Dynamics of nucleosomal DNA

Explore the accessibility and site exposure of nucleosomal DNA for protein binding, revealing cooperative invasion and conformational fluctuations. Understand nucleosome dynamics through FRET systems and site exposure assays. Discover the rates of site exposure and re-wrapping using advanced techniques.

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Dynamics of nucleosomal DNA

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  1. Dynamics of nucleosomal DNA

  2. Felsenfeld, G. & Groudine, M. (2003), Nature 421: 448-453

  3. GCN4; Ellenberger et al., Cell 71: 1223, 1992 NF-KB; Chen et al., Nature 391: 410, 1998 Zif268; Pavletich & Pabo, Science 252: 809, 1991 Bam H1; Newman et al., Science 269: 656, 1995

  4. R + R k12 k23 k21 k32 R k23 + R k32 A model for the spontaneous accessibility of nucleosomal DNA target sites Polach & Widom, 1995

  5. Any protein can bind to DNA in a nucleosome, and we can predict its affinity R + R k12 k23 Kd,nucleosome = Kd,naked DNA / Keqconf k21 k32 R k23 + R k32 Polach & Widom, 1995

  6. Cooperative invasion of a nucleosome by collaborative competition with histone octamer N-X DG01 DG03 DG02 N N-XY N-Y X Y dGXY = -DG01 Polach & Widom, 1996 Miller & Widom, 2003

  7. Uncoiling vs sliding for a mononucleosome Energetic costs are similar Anderson et al., 2002

  8. Test of uncoiling vs sliding for a mononucleosome • Extra DNA favors mobility but does not affect uncoiling Anderson et al., 2002

  9. 1 10–1 Relative Keq 10–2 10–3 Location in nucleosome Left end Right end middle Site exposure occurs without nucleosome sliding Anderson et al., 2002

  10. Site exposure via stepwise unwrapping from one end of the nucleosomal DNA Luger & Richmond, 1997

  11. FRET systems for analysis of nucleosome dynamics Li & Widom, 2004

  12. Artificially stable wrapping in sub-physiological ionic strength • Completely dissociated at > 1.5 M NaCl • Titrate between these to measure Keq in physiological ionic strength Intensity Wavelength (nm) Nucleosome conformational fluctuations in physiological ionic strength solution Acceptor on H2A K119C Li & Widom, 2004

  13. FRET efficiency vs monovalent cation concentration 1 E/E0 0 0 0.5 1 1.5 2 H2A-K119C H3-V35C Monovalent cation concentration (M) Li & Widom, 2004

  14. Equilibrium constant for DNA unwrapping vs monovalent cation concentration 100 10 1 Keq 0.1 H2A-K119C 0.01 H3-V35C 0.001 0.01 0.1 1 10 Monovalent cation concentration (M) Li & Widom, 2004

  15. Cy3 601–147 601–147 LexA L Cy3 35 601–147 LexA L Cy3 57 601–147 Cy3 69 Cy3 fluorescence donor Site exposure at internal nucleosome target sites: Cy3-labeled DNA constructs Hannah Tims

  16. Site exposure at internal nucleosome target sites:Cy5-labeled histone octamers and Cy3-labeled DNA Cy5 H3V35C 69 1 Cy5 H2AK119C 57 Cy5 H4S47C Cy5 H2BT112C Cy5 H4L22C Cy5 H2AA45C 35 Hannah Tims

  17. V35C Dyad Reduced equilibrium constant for site exposure near nucleosome dyad Hannah Tims

  18. R + R k12 k23 k21 k32 R k23 + R k32 Polach & Widom, 1995

  19. 1 8 27 147 LexA147-left: 1 121 140 147 LexA147-right: DNA constructs for LexA protein binding 1 147 601-147: Li & Widom, 2004

  20. 1 121 140 147 Intensity Wavelength (nm) No change in FRET when LexA binds to DNA end opposite fluorescence donor Li & Widom, 2004

  21. 1 8 27 147 Intensity Wavelength (nm) DNA unwrapping detected by FRET when LexA binds near fluorescence donor Li & Widom, 2004

  22. 1 8 27 147 1 0.8 0.6 FRET Efficiency 0.4 0.2 0 0 100 101 102 103 104 105 [LexA] (nM) Nucleosome conformational change driven by LexA binding near fluorescence donor Li & Widom, 2004

  23. Cy3 601–147 Cy3 601–147 LexA L Cy3 601–147 LexA 17 Cy3 601–147 LexA 27 Cy3 fluorescence donor LexA binding site Site exposure at internal nucleosome target sites Hannah Tims

  24. Cy5 acceptor Cy3 donor LexA 7 LexA 17 LexA27 Protein binding to internal DNA target sites Hannah Tims

  25. Sites further inside the nucleosome are less accessible (more costly) for protein binding Hannah Tims

  26. k23 k12 k32 k21 + LexA k12 k21 Two assays for the rates of site exposure and re-wrapping in nucleosomes Stopped-flow FRET FRET-FCS Li, Levitus, Bustamante, & Widom, 2005

  27. A B 1.0 1.0 Fluorescence (a.u.) Fluorescence (a.u.) 0.9 0.9 0.8 0.8 0.7 0.7 Residuals Residuals 0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 0.1 0.1 Time (s) Time (s) 0 0 -0.1 -0.1 0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 Stopped-flow analysis of LexA binding to buried nucleosomal target site Mock reaction (no LexA) 200 nM LexA Li, Levitus, Bustamante, & Widom, 2005

  28. acceptor donor acceptor Nucleosome dynamics analyzed by fluorescence correlation spectroscopy Li, Levitus, Bustamante, & Widom, 2005

  29. Diffusion-only model G (t) FCS analysis of nucleosomes labeled with donor-only Li et al., 2005

  30. A B 0.5 15 0.4 10 Donor Autocorrelation Function 0.3 GDA () / GD () 5 0.2 0 0.1 0.0 -5 0.001 0.01 0.1 1 10 100 1000 0.001 0.01 0.1 1 10 100 1000 lag time (ms) lag time (ms) Donor-acceptor (D–A / D) Ratio function Donor-only Nucleosome dynamics analyzed by fluorescence correlation spectroscopy Li, Levitus, Bustamante, & Widom, 2005

  31. Rapid spontaneous site exposure in nucleosomes ~4 sec–1 > 5x108 M–1 sec–1 (slow) ~20–90 sec–1 + • Explains how remodeling factors can be recruited to particular nucleosomes on a biologically relevant timescale • Sets tight limits to kinetic efficiency in regulatory protein binding • Suggests that the major impediment to polymerase elongation is re-wrapping of the nucleosomal DNA Li, Levitus, Bustamante, & Widom, 2005

  32. Single Nucleosomes k23 k4 k12 + k32 k21 Nucleosome Array k23 k4 k12* + k32 k21* Site exposure in long chains of nucleosomes Michael Poirier

  33. BamH1 HindII HaeIII RsrII PmlI SacI DraI PstI StyI NsiI mp2 Monomer mp2 mp1 Dimer mp2 mp1 mp1 mp1 mp1 mp1 mp1 mp1 mp1 mp1 mp1 mp1 mp1 mp1 mp1 mp1 mp1 Heptadecamer Site exposure in long chains of nucleosomes Michael Poirier

  34. 250 nm 1  Cation-dependent folding of 17-mers analyzed by AFM a b Extended (low [NaCl]) c d Compact (+ Mg2) Poirier, Bussiek, Langowski, & Widom

  35. Nucleosomal site accessibility in a chromatin fiber Heptadecamer mp1 mp1 mp1 mp1 mp1 mp1 mp1 mp1 mp2 mp1 mp1 mp1 mp1 mp1 mp1 mp1 mp1 Dimer mp2 mp1 HaeIII PstI StyI DraI PmlI HindII BamH1 Basepair (in nucleosome) Michael Poirier

  36. Site accessibility in linker DNA 17mer mp1 mp1 mp1 mp1 mp1 mp1 mp1 mp1 mp2 mp1 mp1 mp1 mp1 mp1 mp1 mp1 mp1 Dimer mp2 mp1 SacI NsiI RsrII Keq Basepair (in linker) Michael Poirier

  37. R + R k12 k23 k21 k32 Site exposure equilibrium constants depend on the affinity of histone-DNA interactions Anderson & Widom, 2000 Widom, 2001

  38. Site exposure equilibrium constants depend on the affinity of histone-DNA interactions 5S rRNA gene DNA SELEX Non-natural high affinity DNA Anderson & Widom, 2000 Widom, 2001

  39. R + R k12 k23 k21 k32 Therefore, the equilibrium locations of nucleosomes along DNA depend on the local affinities of histone-DNA interactions Site exposure equilibrium constants depend on the affinity of histone-DNA interactions Anderson & Widom, 2000 Widom, 2001 Segal et al., 2006

  40. Felsenfeld, G. & Groudine, M. (2003), Nature 421: 448-453

  41. Acknowledgements Nucleosome dynamics Peggy Lowary Kevin Polach Jeff Anderson Gu Li Marcia Levitus (Berkeley, Arizona State) Carlos Bustamante (Berkeley) Michael Poirier Karissa Fortney Hannah Tims Georgette Moyle

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