Interactions of Isw2 chromatin remodeling complex with nucleosomal arrays: Analyses using recombinant yeast histones and immobilized templates
Interactions of Isw2 chromatin remodeling complex with nucleosomal arrays: Analyses using recombinant yeast histones and immobilized templates
复制标题
DOI：
10.1128/mcb.21.6.2098-2106.2001
复制
发表时间：
2001-03-01
影响因子：
5.3
通讯作者：
Tsukiyama, T
中科院分区：
文献类型：
--
作者：
Gelbart, ME;Rechsteiner, T;Tsukiyama, T
为了促进染色质相关蛋白在芽殖酵母Saccharomyces cerevisiae的生化表征，我们已经开发了一个系统，组装核小体阵列固定化模板使用重组酵母核心组蛋白。该系统使我们能够分析Isw 2 ATP依赖的染色质重塑复合物与核小体阵列的相互作用。我发现Isw 2复合物以ATP非依赖性方式与裸DNA和核小体阵列有效地相互作用，这表明在这种物理相互作用之后的步骤中需要ATP。我们鉴定了Isw 2复合物的第二个亚基，其由开放阅读框YGL 133 w编码（本文称为ITC1），并且发现复合物的两个亚基Isw 2p和Itc1p对于与DNA和核小体阵列的有效相互作用是必需的。这两个亚基也是核小体刺激的ATP酶活性和Be复合物的染色质重塑活性所必需的。最后，我们发现，ITC1是必不可少的Isw 2，复杂的功能在体内，因为isw 2和ITC1缺失突变体表现出几乎相同的表型。这些结果证明了我们的体外系统在研究染色质相关蛋白和核小体阵列之间的相互作用的效用。
To facilitate the biochemical characterization of chromatin-associated proteins in the budding yeast Sac-charomyces cerevisiae, we have developed a system to assemble nucleosomal arrays on immobilized templates using recombinant yeast core histones. This system enabled us to analyze the interaction of Isw2 ATP-dependent chromatin remodeling complex with nucleosomal arrays. me found that Isw2 complex interacts efficiently with both naked DNA and nucleosomal arrays in an ATP-independent manner, suggesting that ATP is required at steps subsequent to this physical interaction. We identified the second subunit of Isw2 complex, encoded by open reading frame YGL 133w (herein named ITC1), and found that both subunits of the complex, Isw2p and Itc1p, are essential for efficient interaction with DNA and nucleosomal arrays. Both subunits are also required for nucleosome-stimulated ATPase activity and chromatin remodeling activity of Be complex. Finally, we found that ITC1 is essential for function of Isw2, complex in vivo, since isw2 and itc1 deletion mutants exhibit virtually identical phenotypes. These results demonstrate the utility of our in vitro system in studying interactions between chromatin-associated proteins and nucleosomal arrays.