Analysis of the yeast SWI1, SWI2, and SWI3 proteins

我们研究的总体目标是确定染色体结构如何影响基因表达和 转录机器如何与这种结构相抗衡。我们的总体战略一直是专注于 进化上保守的蛋白质复合体SWI/SNF，这是酵母表达子集所必需的 基因和几种转录激活剂的活性。酵母中的遗传学研究表明SWI/SNF 通过对抗染色质介导的转录抑制促进转录，我们的体外 研究表明，~1Mda SWI/SNF复合体可以利用ATP水解产生的能量 动员核小体，破坏核小体结构。我们最近的研究表明，SWI/SNF可能是 在重塑酶中是独一无二的，因为它的作用可以破坏紧密的异染色质，促进 同源重组。这一提议继续利用强大的遗传和生化 在酵母中研究SWI/SNF在体内的作用及其生化机制的机会 其中SWI/SNF在体外破坏常染和异染色质结构。我们的第一个目标是剖析 SWI/SNF重塑异染色质结构的生化机制包括实验 这项测试测试了SWI/SNF是否包含与异染色质成分相互作用的独特亚基。在我们的 第二个目标，我们将研究SWI/SNF在异染色质动力学中是否发挥普遍作用 活着。我们建议测试SWI/SNF是否破坏了异色结构的虚假组装 常染色质基因座，以及SWI/SNF是否促进TY5转座为异染色质。我们的第三个 目的探讨DNA序列和组蛋白乙酰化在调节人类免疫缺陷病毒类型中的作用。 SWI/SNF作用产生的重塑。这一目标涉及染色质生物化学和单分子 接近了。在我们的第四个目标中，我们建议研究SWI/SNF和SWI/SNF的结构- 用EM方法研究核小体复合体。这些研究还包括对含有 亚基-GFP融合以定位SWI/SNF亚基在获得的结构中的位置。

The overall objective of our research is to determine how chromosome structure affects gene expression and how the transcription machinery contends with this structure. Our general strategy has been to focus on an evolutionarily conserved protein complex, SWI/SNF, which is required for expression of a subset of yeast genes and for the activity of several transcriptional activators. Genetic studies in yeast indicate that SWI/SNF facilitates transcription by antagonizing chromatin-mediated transcriptional repression, and our in vitro studies indicate that the ~1 Mda SWI/SNF complex can use the energy derived from ATP hydrolysis to mobilize nucleosomes and disrupt nucleosome structure. Our recent work indicates that SWI/SNF may be unique among remodeling enzymes as its action can disrupt compact heterochromatin, promoting homologous recombination. This proposal continues to exploit the powerful genetic and biochemical opportunities available in yeast to investigate the role of SWI/SNF in vivo and the biochemical mechanism by which SWI/SNF disrupts euchromatic and heterochromatic structures in vitro. Our first objective will dissect the biochemical mechanism by which SWI/SNF remodels heterochromatin structures, including experiments that test whether SWI/SNF contains a unique subunit that interacts with heterochromatin components. In our second objective we will investigate whether SWI/SNF plays a general role in heterochromatin dynamics in vivo. We propose to test if SWI/SNF disrupts the spurious assembly of heterochromatic structures at euchromatic loci, and whether SWI/SNF facilitates Ty5 transposition into heterochromatin. Our third objective will investigate the role of DNA sequence and histone acetylation in regulating the type of remodeling produced due to SWI/SNF action. This aim involves chromatin biochemistry and single molecule approaches. In our fourth aim, we propose to investigate the structure of SWI/SNF and SWI/SNF- nucleosome complexes by EM methodologies. These studies also involve imaging of complexes that harbor subunit-GFP fusions to map locations of SWI/SNF subunits within the obtained structures.