CHARACTERIZATION OF ESSENTIAL PROTEINS INVOLVED IN CHROMOSOME SEGREGATION

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。 正常的细胞分裂需要所有的染色体被复制并平均分离成两个子细胞。在有丝分裂过程中，姐妹染色单体通过被称为粘附素的蛋白质复合体(通过被称为姐妹染色单体凝聚的过程)结合在一起，直到中期到后期的过渡。凝聚力缺陷可通过增加获得或丢失染色体的速率而导致非整倍体细胞(称为染色体不稳定(CIN))。在哺乳动物系统中，CIN可能促进癌症进展，并在各种癌症中表现出来，但其发生机制尚不清楚。发芽酵母酿酒酵母可用于鉴定哺乳动物中与维持基因组完整性和保守性有关的基因。Hieter实验室最近对ts突变资源进行了遗传筛选，发现了三个新的必需基因，这些基因在突变时会导致CIN、凝聚力缺陷和对DNA损伤剂的敏感性。为了充分了解相应蛋白质的生物学功能，该项目的一个主要目标是确定相互作用的蛋白质，这些蛋白质可能产生关于这些蛋白质参与哪个特定生物过程的信息。使用的主要技术将是串联亲和纯化(TAP)，以分离目的蛋白作为完整复合体的一部分，并确定相互作用的伙伴。TAP方法已被证明可用于分离具有足够纯度的生物活性络合物，用于质谱分析和蛋白质鉴定。TAP获得的结果不仅将扩大我们对酵母中蛋白质相互作用网络的了解，还将为进一步表征这些新蛋白质在DNA修复和姐妹染色单体凝聚力建立和/或维持中的作用提供一个起点。

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. Normal cell division requires that all the chromosomes are duplicated and separated equally into the two daughter cells. During mitosis, sister chromatids are held together by protein complexes known as cohesins (by a process known as sister chromatid cohesion), until the metaphase to anaphase transition. Defective cohesion can lead to aneuploid cells by an increased rate of gain or loss of chromosomes (known as chromosome instability (CIN)). In mammalian systems, CIN may contribute to cancer progression and is exhibited by a variety of cancers, yet the mechanism by which it occurs is poorly understood. The budding yeast Saccharomyces cerevisiae has been useful for identifying genes that are involved in maintenance of genomic integrity and conserved in mammals. A recent genetic screen of a Ts mutant resource in the Hieter laboratory yielded three novel essential genes that lead to CIN, cohesion defects and sensitivity to DNA damaging agents when mutated. In order to fully understand the biological function of the corresponding proteins, a primary goal of this project is to identify interacting proteins that may yield information about which specific biological process these proteins are involved in. The primary technique used will be tandem affinity purification (TAP) in order to isolate the proteins of interest as part of intact complexes and to identify interacting partners. The TAP method has been shown to be useful for isolating biologically active complexes of sufficient purity for mass spectrometric analysis and protein identification. The results obtained from TAP will not only expand our knowledge of protein interaction networks in yeast but will also provide a starting point to further characterize the role of these novel proteins in DNA repair and sister chromatid cohesion establishment and/or maintenance.