Dynamics of assembly and disassembly of nucleosomes along a stretched DNA

University of Illinois at Chicago/Northwestern University

Ranjith Padinhateeri

work done with

Jie Yan and John F. Marko

Nucleosome -- basic unit of Chromatin

DNA

Transcription, replication and other in-vivo DNA processing in eukaryotes take place in the context of chromatin.

Molecular Bilogy Of the Cell : Alberts et al

Nucleosomes

Nucleosome~50nm of DNA is wrappedaround histone octamer

(Alberts et al, MBC)

Nucleosome assembly/disassembly

kinetics, under external force f

f

l=150bp

Single molecule experiments measure change in end-to-end distance (approximately 50 nm/nucleosome)We try to understand experiments in xenopus egg extracts (chaperones present) with no ATP

f

DNA

nucleosome

Stalling force = 3.5pN

Experiments show…

<= Assembly : constant force(magnetic tweezer)Yan et al Mol Bio Cell, 2007Fast decay followed by slow tail

Assembly/disassemblyConstant force (Yan et al)

Disassembly -- constant velocityBennink et al (nat. str. bio 2001)

Assembly of hard-core particles

f

l=150bp (~50nm)

New nucleosomes can occupy only the empty space on the DNA. Empty space decreases as nucleosomes assemble. So is the end to end distance. There is a one-to-one relationbetween the empty space and the end-to-end distance

nucleosome

Single molecule experiments measure change in end-to-end distance (approximately 50 nm/nucleosome)Nucleosomes are like hard-core particles(steric interaction)

Simple model: Random sequential adsorption

Randomly place hard-core particles on a lattice(DNA is like a Lattice)

… and measure the empty space on the lattice; at t=0the whole line is empty

As each particle is adsorbed number of empty sites are reducedby a certain amount (in the cae of nucleosome it is about 50nm)

We can measure the empty space and compute the end-to-end length

Model: Adsorption desorption and diffusion of nucleosomes

Hard-core particles of length l (~50 nm)Measure the the total gap length (empty space on the line)Total gap length is related to the end-to-end-distance measured in the experiments (we compute exptly measuredend-to-end extension from this gap length)

adsorbdesorb

diffuse

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ron i

€

roffi

€

rd

l

Nucleosome Diffusion

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D( f ) = D(0).exp −β . f .ΔL( )In egg extract conditions, at f=1pN, D(f)= 3 10-15 cm2/sec(using free energy = 42kBT)

Thermal fluctuations can form “loops” on the nucleosomesand reptation of these loops could lead to repositioning of the nucleosome (Schiessel et al, PRL,. 2001, 2002))

NAP-1 assists Nucleosome sliding : Y-J Park et al, J. Bio. Chem. (2005)

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ΔL =10 bp

(Schiessel et al)

Their model computes the sequence dependent potential Vi in which nucleosomes move around (rates now depend on this potential)

We determine <Vi> = -42 kBT based on experiments (corresponding to stalling of 3.5 pN) We intend to test the role of sequence in explaining the experimental results

Sequence dependence of nucleosome positioningIt is recently proposed that there is sequence dependence in nucleosome positioning (Segal, Widom et al, Nature 2006)

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ron iroffi

= exp − Vi + f .l( )( )

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di→ j

d j→i= exp Vi −V j( )

Given the potential and rates, we use Monte-Carlo simulation to obtain the dynamics of assembly and disassembly

Model for adsorption, desorption and diffusion in this potential

diffuse

off

on

l

Energies (like V) are expressed in units of kBT

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ron i = r0 exp −α . f .l /kBT( )

roffi = r0 exp β Vi + (1−α ). f .l( )[ ]

Jammed! : no more dimers can be added

Randomly place hard-core dimers on a lattice; what happens If there is no sliding and no desorption?

When 147-mers(nucleosomes) are placed on a DNA randomly, the average amount of empty space is aproximately 25 % of thetotal length of the DNA. One needto have desorption/diffusion to get to higher density of nucleosomes.

Dynamics of assembly -- constant force (f=1pN)

Experiment : Yan et al, Mol Bio Cell (2007)Model with D=5 10-15 cm2/secModel with No diffusion (jamming; gap density : 0.25)Inset: zoomed in view -- final filling facilitated by diffusion

• NO ATP• Egg extract

Early time : exponentially fast filling; Late time: reorganization via diffusion --> slow filling

Assembly and disassembly -- constant force

ModelAssembly stalls around 3.5 pNBarrier to unwrap

ExperimentYan et al Mol. Bio. Cell(2007)

determines the contribution of the force into the on-rate (75 %); determine barrier height.

Velocity of one end of the DNA

Dynamics display “memory” of nucleosome configuration

Dynamics of disassembly -- constant velocity

ModelExperimentBennink et al (2001)Nature Str. Biol.

DNA of Lambda phage virusDNA with homogeneous sequence

Sequence is essential to explain the slope in the curve

World-line of nucleosomes -- disassembly - lambda DNA

Nucleosomes on the left part are short lived -- this is due toa specific nature of lambda DNA sequence. This prediction can be tested experimentally.

Each line corresponds toa nucleosome.Line ends whenthe nucleosomeis desorbed fromthe DNA

Conclusion

• A model with adsorption, desorption and diffusion of nucleosomes can explain some of the in vitro experiments

• We can estimate the adsorption-rate, desorption rate and the amount of nucleosome sliding.

• We estimate the dependence of force in the on- and off- rates.

• Sequence effects are important in explaining experiments.