Logie C, Peterson C L
Program in Molecular Medicine and Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, MA 01605, USA.
EMBO J. 1997 Nov 17;16(22):6772-82. doi: 10.1093/emboj/16.22.6772.
A novel, quantitative nucleosome array assay has been developed that couples the activity of a nucleosome 'remodeling' activity to restriction endonuclease activity. This assay has been used to determine the kinetic parameters of ATP-dependent nucleosome disruption by the yeast SWI/SNF complex. Our results support a catalytic mode of action for SWI/SNF in the absence of nucleosome targeting. In this quantitative assay SWI/SNF and ATP lead to a 100-fold increase in nucleosomal DNA accessibility, and initial rate measurements indicate that the complex can remodel one nucleosome every 4.5 min on an 11mer nucleosome array. In contrast to SWI/SNF action on mononucleosomes, we find that the SWI/SNF remodeling reaction on a nucleosome array is a highly reversible process. This result suggests that recovery from SWI/SNF action involves interactions among nucleosomes. The biophysical properties of model nucleosome arrays, coupled with the ease with which homogeneous arrays can be reconstituted and the DNA accessibility analyzed, makes the described array system generally applicable for functional analysis of other nucleosome remodeling enzymes, including histone acetyltransferases.
一种新的定量核小体阵列分析方法已被开发出来，该方法将核小体“重塑”活性与限制性内切酶活性相结合。此分析方法已用于确定酵母SWI/SNF复合物对ATP依赖性核小体破坏的动力学参数。我们的结果支持在没有核小体靶向的情况下SWI/SNF的催化作用模式。在这种定量分析中，SWI/SNF和ATP使核小体DNA可及性增加100倍，初始速率测量表明该复合物在11聚体核小体阵列上每4.5分钟可重塑一个核小体。与SWI/SNF对单核小体的作用相反，我们发现SWI/SNF在核小体阵列上的重塑反应是一个高度可逆的过程。这一结果表明，从SWI/SNF作用中恢复涉及核小体之间的相互作用。模型核小体阵列的生物物理特性，加上均匀阵列易于重构以及DNA可及性易于分析，使得所描述的阵列系统普遍适用于其他核小体重塑酶（包括组蛋白乙酰转移酶）的功能分析。
