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

基本信息

批准号：
7641050
负责人：
Timothy G Formosa
金额：
$ 30.12万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-03-01 至 2011-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7641050
关键词：
ATP Hydrolysis Address Affect Alleles Architecture Binding Biochemical Genetics Cells Chromatin Collaborations Complex Core Protein DNA DNA Polymerase II DNA Restriction Enzymes DNA biosynthesis DNA-Directed DNA Polymerase Funding Gel Genetic Screening Genetic Transcription Genetic screening method Genome Goals Grant Histones In Vitro Individual Life Location Maps Measures Metabolism Methylation Modeling Mutate Mutation Names Nucleosome Core Particle Nucleosomes Pathway interactions Phenotype Play Positioning Attribute Process Property Proteins RNA Relative (related person)Role Saccharomyces cerevisiae Site Structure Taiwan Testing Work Yeasts base chromatin immunoprecipitation chromatin remodeling dimer genetic analysis histone modification in vitro Assay in vitro activity in vivo insight mutant novel nuclease repaired research study role model segregation study characteristics two-dimensional

项目摘要

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.

描述（由申请人提供）：必须将大型真核基因组包装为染色质，以使它们保持有组织和稳定，但这通常会抑制对DNA的可及性。因此，控制转录和DMA复制需要有选择性克服该染色质屏障的能力。我们已经确定了酵母细胞中一种称为YFACT的因素，该因素使用一种新型机制来减少染色质的抑制作用。与使用从ATP水解为重新定位的核小体衍生的能量的更好特征的重塑因子不同，YFACT会改变单个核小体的结构而无需水解ATP或更改核小体的位置。我们称这种新颖的机制为“核小体重组”，以将其与重塑区分开。 YFACT是转录和复制所必需的。该提案的目的是分析核小体重组的机制，并确定这种重组活性如何参与活酵母细胞的转录和复制的各种不同步骤。 YFACT必须与许多不同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表征YFACT与其两个已知伴侣RPA和组蛋白之间的相互作用，然后确定这种结合如何有助于YFACT功能的范围。 RPA是DNA复制的关键因素，组蛋白组成核小体的蛋白质核心，因此这些相互作用可能是YFACT的多种功能的核心。提出和测试这些相互作用如何有助于复制，修复和转录的模型。第二个目的是更好地表征使用纯化成分核小体重组的机理。将使用核酸酶探测重组状态，将测试重组核小体的稳定性，并将彼此映射组件的接近度。最后，将使用染色质免疫沉淀，遗传稳定性测试和2D凝胶来解决YFACT成分中的突变如何影响转录中的关键中间体和活细胞中的复制。制作精确的DNA拷贝的能力，可以作为RNA允许正常的细胞代谢或作为隔离后代的DNA的能力，这在根本上很重要。 YFACT是两个过程的重要组成部分，因此研究它可以同时了解几种不同的中心途径。