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（由申请人提供）：大型真核基因组必须包装为染色质，以保持其有序和稳定，但这通常会抑制 DNA 的可及性。因此，控制转录和 DMA 复制需要能够选择性地克服这种染色质屏障。我们在酵母细胞中发现了一种名为 yFACT 的因子，它使用一种新机制来减弱染色质的抑制作用。与使用 ATP 水解产生的能量重新定位核小体的更好表征的重塑因子不同，yFACT 改变单个核小体的结构，而不水解 ATP 或改变核小体的位置。我们将这种新机制称为“核小体重组”，以区别于重塑。转录和复制都需要 yFACT。该提案的目标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。该提案的首要目标是更好地表征 yFACT 与其 2 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影响 yFACT 的功能范围。 RPA 是 DNA 复制的关键因素，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 多种功能的核心。提出并测试了这些相互作用如何促进复制、修复和转录的模型。第二个目标是更好地表征使用纯化成分的核小体重组机制。将使用核酸酶探测重组状态，测试重组核小体的稳定性，并绘制各成分之间的接近程度。最后，染色质免疫沉淀、遗传稳定性测试和二维凝胶将用于解决 yFACT 成分的突变如何影响活细胞转录和复制的关键中间体。准确复制 DNA 的能力至关重要，无论是作为 RNA 进行正常的细胞代谢，还是作为 DNA 进行分离给后代，这一点至关重要。 yFACT 是这两个过程的重要且新颖的组成部分，因此研究它可以同时深入了解几个不同的中心途径。