Baetz Kristin K, Krogan Nevan J, Emili Andrew, Greenblatt Jack, Hieter Philip
Centre for Molecular Medicine and Therapeutics, Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada V5Z 4H4.
Mol Cell Biol. 2004 Feb;24(3):1232-44. doi: 10.1128/MCB.24.3.1232-1244.2003.
The budding yeast centromere-kinetochore complex ensures high-fidelity chromosome segregation in mitosis and meiosis by mediating the attachment and movement of chromosomes along spindle microtubules. To identify new genes and pathways whose function impinges on chromosome transmission, we developed a genomic haploinsufficiency modifier screen and used ctf13-30, encoding a mutant core kinetochore protein, as the reference point. We demonstrate through a series of secondary screens that the genomic modifier screen is a successful method for identifying genes that encode nonessential proteins required for the fidelity of chromosome segregation. One gene isolated in our screen was RSC2, a nonessential subunit of the RSC chromatin remodeling complex. rsc2 mutants have defects in both chromosome segregation and cohesion, but the localization of kinetochore proteins to centromeres is not affected. We determined that, in the absence of RSC2, cohesin could still associate with chromosomes but fails to achieve proper cohesion between sister chromatids, indicating that RSC has a role in the establishment of cohesion. In addition, numerous subunits of RSC were affinity purified and a new component of RSC, Rtt102, was identified. Our work indicates that only a subset of the nonessential RSC subunits function in maintaining chromosome transmission fidelity.
出芽酵母着丝粒-动粒复合体通过介导染色体沿纺锤体微管的附着和移动,确保有丝分裂和减数分裂中染色体的高保真分离。为了鉴定其功能影响染色体传递的新基因和途径,我们开发了一种基因组单倍体不足修饰筛选方法,并使用编码突变型动粒核心蛋白的ctf13-30作为参考点。我们通过一系列二次筛选证明,基因组修饰筛选是一种成功的方法,可用于鉴定编码染色体分离保真度所需的非必需蛋白的基因。我们筛选中分离出的一个基因是RSC2,它是RSC染色质重塑复合体的一个非必需亚基。rsc2突变体在染色体分离和黏连方面均有缺陷,但动粒蛋白在着丝粒上的定位不受影响。我们确定,在没有RSC2的情况下,黏连蛋白仍可与染色体结合,但无法在姐妹染色单体之间实现适当的黏连,这表明RSC在黏连的建立中起作用。此外,对RSC的多个亚基进行了亲和纯化,并鉴定出RSC的一个新组分Rtt102。我们的工作表明,只有一部分非必需的RSC亚基在维持染色体传递保真度方面发挥作用。