IDENTIFICATION OF COMPONENTS OF THE HIR COMPLEX REQUIRED FOR CHROMATIN ASSEMBLY

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。核小体是真核生物染色体的基本构件，由DNA包裹在组蛋白蛋白上形成。组蛋白在DNA上的快速有序沉积是由染色质组装蛋白介导的。在萌芽酵母中，一组经基因鉴定的四种Hir蛋白(Hir1、Hir2、Hir3和Hpc2)已知与ASF1协同作用，以确保组蛋白四聚体的适当沉积(Sutton等，Genetics，2001；Sharp等，Curr Biol，2001)。HIR蛋白在维持着丝粒的染色质结构方面也有突出的作用，从而有助于准确的染色体分离(Sharp等人，gene Dev，2002)。然而，HIR蛋白复合体的生化性质及其促进染色质正确组装的机制在很大程度上仍未被探索。例如，对HIR蛋白的天然低聚状态几乎一无所知，以前的遗传实验也没有表明是否存在尚未发现的复合体亚单位。因此，我们提出了一种结构/功能的方法，即分离天然的HIR蛋白复合体，并用质谱学和功能分析对其进行表征。

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. Nucleosomes are the fundamental building blocks of the eukaryotic chromosome, formed by wrapping DNA around histone proteins. The rapid and orderly deposition of histones on DNA is mediated by chromatin assembly proteins. In budding yeast, a set of four Hir proteins (Hir1, Hir2, Hir3, and Hpc2) identified genetically are known to act in concert with Asf1 to ensure proper deposition of histone tetramers (Sutton et al., Genetics, 2001; Sharp et al., Curr Biol, 2001). The Hir proteins also have a prominent role in maintaining the chromatin structure at the centromere, thereby contributing to accurate chromosome segregation (Sharp et al., Genes Dev, 2002). However, the biochemical nature of Hir protein complexes and their mechanisms for promoting proper chromatin assembly have remained largely unexplored. For example, almost nothing is known about the native oligomeric state of the Hir proteins, and previous genetic experiments do not indicate whether there may be undiscovered subunits of the complex. We therefore propose a structure/function approach by isolating native Hir protein complexes and characterizing them by both mass spectrometry and functional assays.