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， 转录机制是如何与这种结构竞争的。我们的总体战略是， 进化上保守的蛋白质复合物，SWI/SNF，这是表达一个子集的酵母所必需的。 基因和几种转录激活因子的活性。酵母遗传学研究表明，SWI/SNF 通过拮抗染色质介导的转录抑制来促进转录，我们的体外研究表明， 研究表明，~1 Mda SWI/SNF复合物可以利用ATP水解产生的能量， 移动核小体并破坏核小体结构。我们最近的工作表明，SWI/SNF可能是 在重塑酶中是独特的，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物化学 酵母中可利用的机会来研究SWI/SNF在体内的作用和生化机制， 其中SWI/SNF在体外破坏常染色质和异染色质结构。我们的第一个目标是解剖 SWI/SNF重塑异染色质结构的生化机制，包括实验 测试SWI/SNF是否包含与异染色质组分相互作用的独特亚基。在我们 第二个目的，我们将研究SWI/SNF是否在异染色质动力学中起普遍作用， vivo.我们建议测试SWI/SNF是否会破坏异色结构的虚假组装， 常染色质基因座，以及SWI/SNF是否促进Ty 5转座进入异染色质。我们的第三 目的探讨DNA序列和组蛋白乙酰化在调节细胞凋亡类型中的作用。 由于SWI/SNF作用而产生的重塑。这一目标涉及染色质生物化学和单分子 方法。在我们的第四个目标中，我们建议研究SWI/SNF和SWI/SNF的结构- 核小体复合物的EM方法。这些研究还涉及到对复合物的成像， 亚基-GFP融合物，以绘制SWI/SNF亚基在所获得的结构内的位置。

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.