McQuaid Mary E, Pinder Jordan B, Arumuggam Niroshaathevi, Lacoste Jessica S C, Chew Joyce S K, Dobson Melanie J
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada.
Nucleic Acids Res. 2017 Oct 13;45(18):10518-10533. doi: 10.1093/nar/gkx703.
The yeast 2-μm plasmid is a remarkable genetic parasite, managing efficient maintenance at high-copy number with minimal impact on the host. Equal partitioning of the plasmid upon host cell division requires plasmid proteins Rep1 and Rep2 and the plasmid STB locus. The Rep proteins and the plasmid-encoded Raf protein also regulate plasmid gene transcription. In this study, protein interaction assays, sequence analyses and mutational approaches were used to identify domains and residues in Rep2 and Raf required for association with Rep1 and Rep2 and to delineate the Rep2 DNA-binding domain. Rep2 and Raf displayed similarities in interactions with Rep1 and Rep2, in having Rep1 promote their STB association in vivo, and in stabilizing Rep protein levels. Rep2 mutants impaired for self-association were competent for transcriptional repression while those deficient for Rep1 association were not. Surprisingly, Rep2 mutants impaired for either Rep1 interaction or self-association were able to maintain efficient plasmid inheritance provided Raf was present and competent for Rep protein interaction. Our findings provide insight into the Rep protein complexes required for partitioning and transcriptional repression, and suggest that in addition to its transcriptional function, Raf stabilization of Rep partitioning proteins contributes to the remarkable persistence of the 2-μm plasmid.
酵母2-μm质粒是一种非凡的遗传寄生物，能以高拷贝数高效维持自身，对宿主的影响极小。宿主细胞分裂时质粒的均等分配需要质粒蛋白Rep1和Rep2以及质粒的STB位点。Rep蛋白和质粒编码的Raf蛋白也调控质粒基因转录。在本研究中，利用蛋白质相互作用分析、序列分析和突变方法来鉴定Rep2和Raf中与Rep1和Rep2结合所需的结构域和残基，并描绘Rep2的DNA结合结构域。Rep2和Raf在与Rep1和Rep2的相互作用、Rep1在体内促进它们与STB的结合以及稳定Rep蛋白水平方面表现出相似性。自我结合受损的Rep2突变体能够进行转录抑制，而那些与Rep1结合缺陷的突变体则不能。令人惊讶的是，只要Raf存在且能够与Rep蛋白相互作用，无论是与Rep1相互作用还是自我结合受损的Rep2突变体都能够维持高效的质粒遗传。我们的研究结果为质粒分配和转录抑制所需的Rep蛋白复合物提供了见解，并表明除了其转录功能外，Raf对Rep分配蛋白的稳定作用有助于2-μm质粒的显著持久性。
