Biochemical and Genetic Analysis of yFACT, A Novel Nucleosome Reorganizing Factor

新型核小体重组因子 yFACT 的生化和遗传分析

• 批准号: 7148488
• 负责人: Timothy G Formosa
• 金额: $ 31.02万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7148488
• 关键词: DNA binding protein; DNA replication; SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; chromatin; chromatography; conformation; deoxyribonuclease I; eukaryote; functional /structural genomics; fungal genetics; fungal proteins; gel mobility shift assay; gene interaction; genetic mapping; genetic transcription; genome; immunoprecipitation; nucleosomes; protein structure function

DESCRIPTION (provided by applicant): Large eukaryotic genomes must be packaged as chromatin to keep them organized and stable, but this generally inhibits accessibility to the DNA. Controlling transcription and DMA replication therefore requires the ability to selectively overcome this chromatin barrier. We have identified a factor called yFACT in yeast cells that uses a novel mechanism to diminish the inhibitory effects of chromatin. Unlike the better- characterized remodeling factors that use energy derived from ATP hydrolysis to reposition nucleosomes, yFACT changes the structure of individual nucleosomes without hydrolysing ATP or changing the location of the nucleosomes. We call this novel mechanism "nucleosome reorganization" to distinguish it from remodeling. yFACT is required for both transcription and replication. The goal of this proposal is to analyze the mechanism of nucleosome reorganization and to determine how this reorganizing activity participates in a variety of distinct steps in transcription and replication in living yeast cells. yFACT must interact with many different proteins to accomplish these diverse functions. The first aim of the proposal is to better characterize the interaction between yFACT and 2 of its known partners, RPA and histones, and then to determine how this binding contributes to the range of yFACT functions. RPA is a key factor in DNA replication and histones compose the protein core of nucleosomes, so these interactions are likely to be central to the diverse functions of yFACT. Models for how these interactions contribute to replication, repair, and transcription are proposed and tested. The second aim is to better characterize the mechanism of nucleosome reorganization using purified components. The reorganized state will be probed using nucleases, the stability of reorganized nucleosomes will be tested, and the proximity of components to one another will be mapped. Finally, chromatin immunoprecipitation, tests of genetic stability, and 2-D gels will be used to address how mutations in yFACT components affect key intermediates in transcription and replication in living cells. The ability to make accurate copies of DNA, either as RNA to allow normal cellular metabolism or as DNA for segregation to progeny, is fundamentally important. yFACT is a vital and novel component of both processes, so studying it provides insight into several distinct central pathways simultaneously.

描述(申请人提供)：大的真核基因组必须包装成染色质，以保持它们的组织性和稳定性，但这通常会阻碍DNA的获取。因此，控制转录和DMA复制需要选择性地克服这一染色质障碍的能力。我们已经在酵母细胞中发现了一种名为yFACT的因子，它使用一种新的机制来减弱染色质的抑制作用。与特征更好的重塑因子不同，yFACT使用来自ATP水解的能量来重新定位核小体，它改变了单个核小体的结构，而不需要水解ATP或改变核小体的位置。我们将这种新的机制称为“核小体重组”，以区别于重塑。转录和复制都需要yFACT。这项建议的目的是分析核小体重组的机制，并确定这种重组活动如何参与活酵母细胞中转录和复制的各种不同步骤。YFACT必须与许多不同的蛋白质相互作用才能完成这些不同的功能。该提案的第一个目的是更好地描述yFACT与其两个已知伙伴RPA和组蛋白之间的相互作用，然后确定这种结合如何对yFACT的一系列功能做出贡献。RPA是DNA复制的关键因子，组蛋白构成核小体的蛋白质核心，因此这些相互作用可能是yFACT各种功能的中心。提出了这些相互作用如何有助于复制、修复和转录的模型，并进行了测试。第二个目的是利用纯化的成分更好地描述核小体重组的机制。重组状态将使用核酸酶来探测，重组核小体的稳定性将被测试，并将绘制组件彼此接近的地图。最后，染色质免疫沉淀、遗传稳定性测试和二维凝胶将被用来解决yFACT组件的突变如何影响活细胞中转录和复制的关键中间产物。准确复制DNA的能力是至关重要的，既可以作为RNA来实现正常的细胞代谢，也可以作为DNA用于后代的分离。YFACT是这两个过程的一个重要而新颖的组成部分，因此研究它可以同时洞察几个不同的中央通路。