Stability of a Human SWI-SNF Remodeled Nucleosomal Array

Abstract

SWI-SNF alters DNA-histone interactions within a nucleosome in an ATP-dependent manner. These alterations cause changes in the topology of a closed circular nucleosomal array that persist after removal of ATP from the reaction. We demonstrate here that a remodeled closed circular array will revert toward its original topology when ATP is removed, indicating that the remodeled array has a higher energy than that of the starting state. However, reversion occurs with a half-life measured in hours, implying a high energy barrier between the remodeled and standard states. The addition of competitor DNA accelerates reversion of the remodeled array by more than 10-fold, and we interpret this result to mean that binding of human SWI-SNF (hSWI-SNF), even in the absence of ATP hydrolysis, stabilizes the remodeled state. In addition, we also show that SWI-SNF is able to remodel a closed circular array in the absence of topoisomerase I, demonstrating that hSWI-SNF can induce topological changes even when conditions are highly energetically unfavorable. We conclude that the remodeled state is less stable than the standard state but that the remodeled state is kinetically trapped by the high activation energy barrier separating it from the unremodeled conformation.

ACKNOWLEDGMENTS

We thank J. Workman and K. Neely from Pennsylvania State University for providing the plasmid p2085S-G5E4 (32), which was used to make the template for the linear array used in Fig.7. We thank N. Francis from our laboratory for labeling the linear template and assembling it into a nucleosomal array. We also appreciate critical reading of the manuscript by N. Francis, A. Saurin, G. Schnitzler, L. Corey, K. Lee, and others from our laboratory, who provided many insightful comments. In addition, we are grateful to M. Hirschel and C. Varughese from Cellex Biosciences, Inc., for growing the Ini-1 cell line used to purify hSWI-SNF.

This work was supported by grants from the NIH (to R.E.K.) and the Damon Runyon-Walter Winchell Foundation (to G.J.N.).