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Vol. 18, Issue 2, 464-474, February 2007

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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^¶

*Department of Physics, University of Illinois at Chicago, Chicago, IL 60607-7059; Department of Physics, National University of Singapore, Singapore 117542; Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; Department of Biochemistry, University of Wisconsin, Madison, WI 53706; ^||Institute of Clinical Chemistry and Laboratory Diagnostics, Heinrich-Heine-University, Medical School, D-40225 Duesseldorf, Germany; and ^¶Departments of Biochemistry, Molecular Biology, Cell Biology, and Physics and Astronomy, Northwestern University, Evanston, IL 60208

Submitted September 11, 2006; Revised November 1, 2006; Accepted November 2, 2006
Monitoring Editor: Kerry Bloom

We have studied assembly of chromatin using Xenopus egg extracts and single DNA molecules held at constant tension by using magnetic tweezers. In the absence of ATP, interphase extracts were able to assemble chromatin against DNA tensions of up to 3.5 piconewtons (pN). We observed force-induced disassembly and opening–closing fluctuations, indicating our experiments were in mechanochemical equilibrium. Roughly 50-nm (150-base pair) lengthening events dominated force-driven disassembly, suggesting that the assembled fibers are chiefly composed of nucleosomes. The ATP-depleted reaction was able to do mechanical work of 27 kcal/mol per 50 nm step, which provides an estimate of the free energy difference between core histone octamers on and off DNA. Addition of ATP led to highly dynamic behavior with time courses exhibiting processive runs of assembly and disassembly not observed in the ATP-depleted case. With ATP present, application of forces of 2 pN led to nearly complete fiber disassembly. Our study suggests that ATP hydrolysis plays a major role in nucleosome rearrangement and removal and that chromatin in vivo may be subject to highly dynamic assembly and disassembly processes that are modulated by DNA tension.

Address correspondence to: John F. Marko (john-marko{at}northwestern.edu)

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