Methods in Molecular Biology Volume 833, 2012, pp 351-371

Preparation of Nucleosomes Containing a Specific H2A–H2A Cross-Link Forming a DNA-Constraining Loop Structure

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ATP-dependent chromatin-remodeling complexes use the energy of ATP hydrolysis to alter nucleosome structure, increase the accessibility of trans-acting factors, and induce nucleosome movement on the nucleosomal DNA. Recent studies suggest that bulge propagation is a major component of the mechanism for SWI/SNF remodeling. We describe in detail a method to prepare a mononucleosomal substrate in which the two H2A N-terminal tails are cross-linked in an intranucleosomal fashion, forming a closed loop around the two superhelical winds of DNA. This substrate is useful for researchers who wish to test processes in which the DNA is transiently or permanently lifted off the histone surface, such as in the bulge propagation model. Our method allows assessment of the extent of cross-linking within the population of nucleosomes used in small-scale experiments, such as assays to test SWI/SNF-remodeling activities.