Analysis of yeast chromatin structure and function

酵母染色质结构和功能分析

• 批准号: 7033550
• 负责人: Craig L Peterson
• 金额: $ 31.61万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7033550
• 关键词: analytical ultracentrifugation; cell cycle; chromatin; fungal genetics; gene expression; gene mutation; genetic regulation; genetic transcription; histones; immunofluorescence technique; immunoprecipitation; intermolecular interaction; nucleosomes; protein folding; protein protein interaction; protein structure function; structural biology

DESCRIPTION (provided by applicant): Our overall objective is to determine how chromosome structure controls the repair of DMA double strand breaks (DSBs) by homologous recombination and how the repair machinery contends with this structure. Recombinational repair of a DSB requires members of the Rad52 group of proteins (Rad51p, Rad52p, Rad54p, RadSO, Mre11p, Xrs2p, Rad55p, Rad57p, Rad59p), and recent biochemical studies have shown that Rad54p has ATP-dependent chromatin remodeling activity. An intact recombinational repair system is essential for genome stability, and inactivation of mammalian Rad54 leads to shortened telomeres and development of cancers. Our general strategy is to use a powerful combination of biochemical and yeast molecular genetic approaches to dissect the dynamics of yeast chromatin structure during the repair of DSBs. The first objective is to investigate changes in chromatin structure that occur following DSB formation and during DMA strand invasion in yeast. These studies will exploit Chromosome Conformation Capture (3C) analysis to test whether phosphorylation of histone H2AX alters chromatin structure surrounding a DSB. In addition, HO endonuclease will be used to create a single DNA double strand break in a set of yeast strains that harbor a novel, homologous donor sequence embedded between positioned nucleosomes. In the second aim biochemical studies are described to dissect the mechanism by which Rad54p facilitates invasion of a Rad51 p nucleoprotein filament into a nucleosomal donor. The third objective describes in vivo and in vitro studies that focus on the roles of the InoSO and Swr1 chromatin remodeling complexes in controlling the dynamics of histone H2AX phosphorylation during checkpoint adaptation and DSB repair. The fourth aim will investigate the role of a novel casein kinase 2-dependent phosphorylation of histone H4 Serine 1 in DSB repair.

描述(申请人提供)：我们的总体目标是确定染色体结构如何通过同源重组控制DNA双链断裂(DSB)的修复，以及修复机制如何与这种结构竞争。DSB的重组修复需要Rad52家族的成员(Rad51p、Rad52p、Rad54p、RadSO、Mre11p、Xrs2p、Rad55p、Rad57p、Rad59p)，最近的生化研究表明Rad54p具有依赖于ATP的染色质重塑活性。完整的重组修复系统对基因组的稳定至关重要，哺乳动物RAD54的失活会导致端粒缩短和癌症的发生。我们的总体策略是使用生化和酵母分子遗传学方法的强大组合来剖析DSB修复过程中酵母染色质结构的动态变化。第一个目标是研究酵母中DSB形成和DMA链入侵过程中染色质结构的变化。这些研究将利用染色体构象捕获(3C)分析来测试组蛋白H2AX的磷酸化是否改变了DSB周围的染色质结构。此外，HO内切酶将被用来在一组酵母菌株中产生单个DNA双链断裂，这些酵母菌株在定位的核小体之间嵌入了新的、同源的供体序列。在第二个目标中，描述了生化研究，以剖析Rad54p促进RAD51p核蛋白细丝侵入核小体供体的机制。第三个目标描述了体内和体外的研究，重点是InoSO和Swr1染色质重塑复合体在检查点适应和DSB修复过程中控制组蛋白H2AX磷酸化动态中的作用。第四个目的是研究组蛋白H4丝氨酸1依赖于酪蛋白激酶2的新的磷酸化在DSB修复中的作用。