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 wrapped around 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, 2007 Fast decay followed by slow tail Assembly/disassembly Constant force (Yan et al) Disassembly -- constant velocity Bennink 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 relation between 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=0 the whole line is empty As each particle is adsorbed number of empty sites are reduced by 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 measured end-to-end extension from this gap length) adsorb desorb diffuse l

Nucleosome Diffusion In egg extract conditions, at f=1pN, D(f)= cm 2 /sec (using free energy = 42k B T) Thermal fluctuations can form “loops” on the nucleosomes and 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) =10 bp (Schiessel et al)

Their model computes the sequence dependent potential V i in which nucleosomes move around (rates now depend on this potential) We determine = -42 k B T 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 positioning It is recently proposed that there is sequence dependence in nucleosome positioning (Segal, Widom et al, Nature 2006)

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 k B T

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 need to 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= cm 2 /sec Model 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 Model Assembly stalls around 3.5 pN Barrier to unwrap Experiment Yan 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 Model Experiment Bennink et al (2001) Nature Str. Biol. DNA of Lambda phage virus DNA 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 to a specific nature of lambda DNA sequence. This prediction can be tested experimentally. Each line corresponds to a nucleosome. Line ends when the nucleosome is desorbed from the 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.