Micromanipulation Studies of Chromatin Fibers

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Micromanipulation Studies of Chromatin Fibers in
Xenopus Egg Extracts Reveal ATP-dependent Chromati
n 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.
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Chromatin Assembly

• l 50 nm for one nucleosome (150 bp)

• Can occur with or without ATP

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Magnetic Tweezers/Near-Field-Magnetic Tweezers
Biotin
Chromatin (DNA Histones)
Streptavidin
Naked DNA (l phage DNA)
Deoxygenin
Anti-deoxygenin
or nonmagnetic bead
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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

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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

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Strategy 2 Near-Field-Magnetic Tweezers
Magnet
97 kb fiber, 1.5 pN
Yan et al preprint 2005
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-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)

Contour lenth (l0) length of polymer when
no force is applied to it
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-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

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-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

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-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

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/-ATP Chromatin Disassembly Near-Field-Magnetic
Tweezer Method (Horizontal)
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-ATP vs. ATP Chromatin Assembly
-ATPLogarithmic Assembly
ATPLinear Assembly
97 kb
Different time scales
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ATP Chromatin Assembly

• Higher temporal resolution of (a), 3 min
• after last time point in (a)
• long runs of assembly and disassembly

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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.
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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)