Analysis of yeast chromatin structure and function

我们的总体目标是确定染色体结构如何控制DMAdouble链的修复 由同源重组引起的断裂(DSB)以及修复机械如何与这种结构相抗衡。 DSB的重组修复需要Rad52蛋白质组的成员(Rad51p，Rad52p， Rad54p、RadSO、Mre11p、Xrs2p、Rad55p、Rad57p、Rad59p)和最近的生化研究表明 Rad54p具有依赖于ATP的染色质重塑活性。一个完整的重组修复系统是 对基因组稳定是必不可少的，哺乳动物RAD54的失活会导致端粒缩短和 癌症的发展。我们的总体策略是使用生化和酵母菌的强大组合 用分子遗传学方法研究酵母染色质结构在修复过程中的动态变化 DSB。第一个目标是研究DSB后染色质结构的变化 在酵母中形成和侵入DMA链。这些研究将利用染色体构象。 捕获(3C)分析以测试组蛋白H_2AX的磷酸化是否改变染色质结构 围绕着一个DSB。此外，HO内切酶将被用来产生单个DNA双链断裂 在一组酵母菌株中，这些酵母菌株之间嵌入了一种新的同源供体序列 核小体。在第二个目标中，描述了生化研究，以剖析其机制。 Rad54p促进Rad51p核蛋白细丝侵入核小体供体。第三 目的描述有关InoSO和Swr1染色质作用的体内和体外研究 重塑复合体在检查点控制组蛋白H_2AX磷酸化动态中的作用 适应和DSB修复。第四个目标是研究一种新的酪蛋白激酶2依赖性蛋白的作用。 组蛋白H4丝氨酸1在DSB修复中的磷酸化。

Our overall objective is to determine how chromosome structure controls the repair of DMAdouble 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 DMAstrand 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 Rad51p 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.