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

描述（由申请人提供）：大型真核生物基因组必须包装成染色质以保持其组织和稳定，但这通常会抑制DNA的可及性。因此，控制转录和DMA复制需要有选择性地克服这种染色质屏障的能力。我们已经在酵母细胞中发现了一个叫做yFACT的因子，它使用一种新的机制来减少染色质的抑制作用。与利用ATP水解产生的能量来重新定位核小体的重构因子不同，yFACT在不水解ATP或改变核小体位置的情况下改变单个核小体的结构。我们称这种新机制为“核小体重组”，以区别于重塑。转录和复制都需要yFACT。本研究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酵母活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。该提案的第一个目标是更好地描述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影响yFACT功能的范围。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。这些相互作用如何促进复制、修复和转录的模型被提出和测试。第二个目的是利用纯化的组分更好地表征核小体重组的机制。重组状态将用核酸酶探测，重组核小体的稳定性将被测试，组分之间的接近度将被绘制。最后，染色质免疫沉淀、遗传稳定性测试和2-D凝胶将用于解决yFACT成分突变如何影响活细胞中转录和复制的关键中间体。无论是作为RNA进行正常的细胞代谢，还是作为DNA分离给后代，精确复制DNA的能力都是至关重要的。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洞察几个不同的中心路径。

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 two 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.