Structural Basis for RCC1 Directed Recruitment of Ran GTPase to Chromatin

项目概要/摘要 真核细胞必须能够在其核与核之间定向运输大分子， 细胞质，并通过有丝分裂分裂细胞。这些基本过程是通过定位 胞质内GDP或GTP结合状态的小Ran鸟苷三磷酸酶（GTdR）蛋白，或 细胞核，并通过在染色体周围产生RanGTP的梯度。空间 RanGTP在细胞核中的定位是通过染色质结合的RCC 1（染色体调节因子）实现的。 缩合）蛋白质。RCC 1将Ran招募到染色体上，并促进RanGDP与 RanGTP，从而在染色体周围产生高浓度的RanGTP。 我们目前缺乏对RCC 1如何与核小体结合以及RCC 1如何与核小体结合的分子理解。 尽管这些相互作用对于基本的细胞功能至关重要， 流程.因此，我们的总体目标是开发描述RCC 1和Ran如何结合的原子模型。 核小体核心颗粒我们的具体目标是： 1.定义RCC 1如何通过生物化学方法与核小体结合。我们将挑战结构 RCC 1如何通过下拉，生物层干涉测量和 荧光共振能量转移实验。 2.确定RCC 1/核小体复合物的结构。我们将使用单晶的 RCC 1/核小体复合物，我们已经生长以确定复合物的结构。这些晶体学 研究工作还将辅以小角度X射线和中子散射实验，以提供解决方案 复杂的结构。 3.确定染色质结合的RCC 1如何结合并激活Ran。我们将测试模型 Ran/RCC 1/核小体复合物，通过分析定向突变对Ran与 RCC 1/核小体复合物和在RCC 1存在下Ran的核苷酸交换活性， 核小体

Project Summary/Abstract A eukaryotic cell must be able to transport macromolecules directionally between its nucleus and cytoplasm, and to divide the cell through mitosis. These fundamental processes are controlled by localizing the small Ran guanosine triphosphatase (GTPase) protein in its GDP or GTP bound state within the cytoplasm or the nucleus respectively, and by generating a gradient of RanGTP around the chromosomes. The spatial localization of RanGTP in the nucleus is achieved through chromatin bound RCC1 (regulator of chromosomal condensation) protein. RCC1 recruits Ran to the chromosomes and promotes the exchange of RanGDP for RanGTP, thereby creating a high concentration of RanGTP around chromosomes. We currently lack a molecular understanding of how RCC1 binds to the nucleosome and how RCC1 recruits Ran to the nucleosome, despite the critical importance of these interactions for basic cellular processes. Our overall goal is therefore to develop atomic models which describe how RCC1 and Ran bind to the nucleosome core particle. Our specific aims are: 1. Define how RCC1 binds to nucleosomes through biochemical methods. We will challenge structural models for how RCC1 interacts with the nucleosome through pulldown, biolayer interferometry and fluorescence resonance energy transfer experiments. 2. Determine the structure of the RCC1/nucleosome complex. We will use single crystals of the RCC1/nucleosome complex we have grown to determine the structure of the complex. These crystallographic studies will be complemented with small angle X-ray and neutron scattering experiments to provide a solution structure of the complex. 3. Determine how chromatin-bound RCC1 binds to and activates Ran. We will test models for the Ran/RCC1/nucleosome complex by analyzing the effects of directed mutations on binding of Ran to the RCC1/nucleosome complex and on Ran's nucleotide exchange activity in the presence of RCC1 and the nucleosome.