Structural Basis for RCC1 Directed Recruitment of Ran GTPase to Chromatin

描述（由申请人提供）：真核细胞必须能够在细胞核和细胞质之间定向运输大分子，并通过有丝分裂分裂细胞。这些基本过程是通过在细胞质或细胞核内分别定位于GTP结合状态或GTP结合状态的小核糖核酸鸟苷三磷酸酶（GTPase）蛋白，并通过在染色体周围产生RanGTP梯度来控制的。RanGTP在细胞核中的空间定位是通过染色质结合的RCC1（染色体凝聚调节因子）蛋白实现的。RCC1将Ran招募到染色体上，并促进RanGDP与RanGTP的交换，从而在染色体周围产生高浓度的RanGTP。尽管这些相互作用对基本细胞过程至关重要，但我们目前对RCC1如何与核小体结合以及RCC1如何将Ran招募到核小体缺乏分子理解。因此，我们的总体目标是建立描述RCC1和Ran如何与核小体核心粒子结合的原子模型。我们的具体目标是：1。通过生化方法定义RCC1如何与核小体结合。我们将通过下拉、生物层干涉和荧光共振能量转移实验来挑战RCC1与核小体相互作用的结构模型。2. 测定RCC1/核小体复合物的结构。我们将使用我们培养的RCC1/核小体复合体的单晶来确定复合体的结构。这些晶体学研究将辅以小角度x射线和中子散射实验，以提供复合物的溶液结构。3. 确定染色质结合的RCC1如何结合并激活Ran。我们将通过分析定向突变对Ran与RCC1/核小体复合物结合的影响，以及在RCC1和核小体存在下Ran的核苷酸交换活性的影响，来测试Ran/RCC1/核小体复合物的模型。公共卫生相关性：当一个细胞分裂时，每个子细胞必须获得等量的携带细胞遗传蓝图的染色体。染色体分布不均或不正确会导致遗传不稳定和癌症。我们的研究方向是通过为真核细胞创建GPS或基因组定位系统来可视化细胞分裂过程中调节染色体均匀分布的分子。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: When a cell divides, each daughter cell must receive an equal share of the chromosomes which carry the cell's genetic blueprint. Unequal or improper distribution of the chromosomes can result in genetic instabilities and cancer. Our studies are directed at visualizing the molecules which regulate the equal distribution of chromosomes during cell division by creating a GPS or genome-positioning system for a eukaryotic cell.