CHARACTERIZATION OF ESSENTIAL PROTEINS INVOLVED IN CHROMOSOME SEGREGATION

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 正常的细胞分裂要求所有的染色体都被复制并平均地分成两个子细胞。在有丝分裂过程中，姐妹染色单体通过蛋白质复合物被称为凝聚素（通过称为姐妹染色单体凝聚的过程）保持在一起，直到中期到后期的转变。 缺陷的凝聚力可通过增加染色体的获得或丢失率（称为染色体不稳定性（CIN））而导致非整倍体细胞。 在哺乳动物系统中，CIN可能有助于癌症进展，并表现为多种癌症，但其发生的机制知之甚少。 芽殖酵母酿酒酵母（Saccharomyces cerevisiae）已用于鉴定参与维持基因组完整性并在哺乳动物中保守的基因。 最近在Hieter实验室对Ts突变资源进行的遗传筛选产生了三个新的必需基因，这些基因在突变时导致CIN、内聚缺陷和对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，该项目的主要目标是鉴定相互作用的蛋白质，这些蛋白质可能会产生有关这些蛋白质参与哪些特定生物过程的信息。 所使用的主要技术将是串联亲和纯化（TAP），以分离作为完整复合物的一部分的感兴趣的蛋白质并鉴定相互作用的伴侣。 TAP方法已被证明可用于分离具有足够纯度的生物活性复合物，用于质谱分析和蛋白质鉴定。 从TAP获得的结果将不仅扩大我们的知识，蛋白质相互作用网络在酵母，但也将提供一个起点，进一步表征这些新的蛋白质在DNA修复和姐妹染色单体凝聚力的建立和/或维护的作用。

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.