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DNA双链的修复 断裂（DSB）的同源重组，以及修复机制如何与这种结构竞争。 DSB的免疫修复需要Rad52组蛋白的成员（Rad51p，Rad52p， Rad54p、RadSO、Mre11p、Xrs2p、Rad55p、Rad57p、Rad59p），并且最近的生物化学研究已经表明， Rad54p具有ATP依赖的染色质重塑活性。完整的重组修复系统是 对于基因组稳定性至关重要，哺乳动物Rad54的失活导致端粒缩短， 癌症的发展。我们的总体策略是使用生化和酵母的强大组合 分子遗传学方法来剖析酵母染色质结构的动态修复过程中， DSB。第一个目的是研究DSB后染色质结构的变化 形成和在酵母中的DMAstrand入侵期间。这些研究将利用染色体构象 捕获（3C）分析，以测试组蛋白H2AX的磷酸化是否改变染色质结构 围绕着一个DSB。此外，HO内切酶将用于产生单个DNA双链断裂 在一组酵母菌株中，所述酵母菌株具有嵌入在定位的DNA之间的新的同源供体序列， 核小体在第二个目标中，描述了生物化学研究，以剖析 Rad54p促进Rad51p核蛋白丝侵入核小体供体。第三 目的描述了体内和体外研究，重点是InoSO和Swr 1染色质的作用 重塑复合物在控制组蛋白H2AX磷酸化的动力学过程中的检查点 适应和DSB修复。第四个目标是研究一种新的酪蛋白激酶2依赖性蛋白激酶的作用。 DSB修复中组蛋白H4丝氨酸1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.