1 / 15

Micromanipulation Studies of Chromatin Fibers in Xenopus Egg Extracts Reveal ATP-dependent

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:

keiko-suarez
Download Presentation

Micromanipulation Studies of Chromatin Fibers in Xenopus Egg Extracts Reveal ATP-dependent

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

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

  3. Magnetic bead Force-induced stretched DNA Coverslip surface Magnetic Tweezers/Near-Field-Magnetic Tweezers Biotin Chromatin (DNA + Histones) Streptavidin Naked DNA (l phage DNA) Deoxygenin Anti-deoxygenin 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 Magnet 97 kb fiber, 1.5 pN Yan et al preprint 2005

  7. -ATP Chromatin Assembly Magnetic Tweezer Method (Vertical) 1 pN • 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) Contourlenth (l0) – length of polymer when no force is applied to it

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

  9. -ATP Chromatin Disassembly Near-Field-Magnetic Tweezer Method (Horizontal) 49 kb fiber • 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. -ATP vs. +ATP Chromatin Assembly -ATP—Logarithmic Assembly +ATP—Linear Assembly 97 kb *Different time scales

  13. +ATP Chromatin Assembly • 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)

More Related