1. Micromanipulation Studies of Chromatin Fibers in Xenopus Egg Extracts Reveal ATP-dependent Chromatin Assembly Dynamics Jie Yan, Thomas J. Maresca, Dunja Skoko, Christian D. Adams, Botao Xiao, Morten O. Christensen, Rebecca Heald, and John F. Marko Goals: 1) To study the assembly and disassembly of chromatin under ATP conditions so as to measure the lengths forces and free energy associated with chromatin. 2) To determine the principal differences between the +ATP and –ATP reactions.

2. l = 50 nm for one nucleosome (~150 bp) Chromatin Assembly Can occur with or without ATP

3. Force-induced stretched DNA Magnetic bead Coverslip surface Deoxygenin Anti-deoxygenin Streptavidin Biotin Chromatin (DNA + Histones) Naked DNA  phage DNA) Magnetic Tweezers/Near-Field-Magnetic Tweezers or nonmagnetic bead

4. Strategy #1: Classic Magnetic Tweezers Vertical Imaging (Dark Field) Advantage: Low-force, low noise measurements Disadvantages: Lower resolution Slow acquisition time Single end imaging only

5. Strategy #2: Near-Field-Magnetic Tweezers Horizontal Imaging Advantages: Finer extension resolution High acquisition rate Simultaneous acquisition of both ends of DNA strand Disadvantages: Not great for low force experiments

6. Strategy #2: Near-Field-Magnetic Tweezers 97 kb fiber, 1.5 pN Magnet Yan et al preprint 2005

7. -ATP Chromatin Assembly Magnetic Tweezer Method (Vertical) 1 pN Contour lenth ( l 0 ) – length of polymer when no force is applied to it (a)2 regimes of chromatin assembly (b) Assembly kinetics independent of length of fiber (c) 3.5 pN force on fiber causes assembly/disassembly equilibrium (stall force)

8. -ATP Chromatin Assembly Near-Field-Magnetic Tweezer Method (Horizontal) (a)No extract (noise) (b)Extract added, assembly with “steps” (c)Force increased to 3.5 pN (stall force), assembly/disassembly equilibrium 79 kb fiber

9. -ATP Chromatin Disassembly Near-Field-Magnetic Tweezer Method (Horizontal) 49 kb fiber (d)Assembly at 1pN, then force increased to 4.5 pN; “steps” of 50 nm observed (e) Assembly at 1pN, then force increased to 15 pN; “steps” of 50 and 100 nm observed

10. -ATP Chromatin Disassembly Near-Field-Magnetic Tweezer Method (Horizontal) 15 kb 49 kb steps (f) Better step resolution using shorter fiber Less noise Plateau durations increased due to fewer nucleosomes on fiber (e) Majority of steps of 50 nm length

11. +/-ATP Chromatin Disassembly Near-Field-Magnetic Tweezer Method (Horizontal)

12. 97 kb -ATP—Logarithmic Assembly+ATP—Linear Assembly *Different time scales -ATP vs. +ATP Chromatin Assembly

13. +ATP Chromatin Assembly (b)Higher temporal resolution of (a), 3 min after last time point in (a) *long runs of assembly and disassembly

14. +ATP Chromatin Assembly As you increase force on the fiber above ~1 pN, net disassembly occurs with runs of assembly and disassembly. Above a force of ~4.2 pN, long runs are all but eliminated.

15. Conclusions Chromatin assembles onto naked DNA in cytoplasmic extracts in the absence of ATP. Initial assembly of nucleosomes onto DNA by the -ATP reaction is not processive. (logarithmic) Stall force of -ATP assembly is estimated to be 3.5 pN. Study indicates that assembly of nucleosomes onto DNA by the +ATP reaction is processive. (linear)