USE OF YTH TO IDENTIFY INTERACTIONS WITH CENTROMERIC NUCLEOSOMES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 真核生物基因组被包装成核小体，核小体是由包裹在组蛋白周围的DNA组成的颗粒。着丝粒是染色质的特殊区域，在有丝分裂过程中充当着丝粒的连接点。在最近的工作过程中，有人提出着丝粒组蛋白核心的结构与典型的组蛋白核心的结构有根本的不同。典型的核小体含有两个分子，分别是组蛋白H_2A、H_2B、H_3和H_4，它们共同构成一个八聚体。然而，最近获得的实验证据表明，着丝粒核小体是一个较小的颗粒，最有可能包含组蛋白H_2A、H_2B、CenH_3和H_4的单一拷贝，H_2A、H_2B、CenH_3和H_4是半体。半体模型预测，含有CenH3的颗粒上没有暴露在正则颗粒上的新表面可以被动粒蛋白特异性结合。利用标准核小体的晶体结构作为起点，我们已经确定了H_2B和CenH_3的区域，这些区域可能暴露在半体中的溶剂中，从而可以被动粒蛋白结合。我们希望使用酵母双杂交系统来筛选这些蛋白的相互作用伙伴，并以组蛋白H4为对照。我们希望识别具有已知动粒功能的蛋白质，这将检验我们的模型，并允许我们对内部动粒结构提出新的假设。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The eukaryotic genome is packaged into nucleosomes, particles that consist of DNA wrapped around histone proteins. Centromeres are specialized regions of chromatin that serve as points of attachment of kinetochore during mitosis. In the course of recent work it has been proposed that the structure of the centromeric histone core is fundamentally different than that of the canonical one. Canonical nucleosomes contain two molecules each of histones H2A, H2B, H3 and H4 that together comprise an octamer. However, recently obtained experimental evidence suggests that centromeric nucleosome is a smaller particle, most likely containing a single copy of histones H2A, H2B, CenH3 and H4, a hemisome. The hemisome model predicts that novel surfaces on the CenH3-containing particle that are not exposed on the canonical particle can be specifically bound by kinetochore proteins. Using the crystal structure of the canonical nucleosome as a starting point we have identified regions of H2B and CenH3 that might be exposed to solvent in a hemisome and thus be available for binding by kinetochore proteins. We would like to use the Yeast Two Hybrid system to screen for interaction partners of these proteins, using histone H4 as a control. We hope to identify proteins with known kinetochore function that will test our model and allow us to propose new hypotheses for the inner kinetochore structure.