Department of Microbiology and Immunology, W. R. Hearst Microbiology Research Center, Weill Medical College of Cornell University, 1300 York Ave., New York, New York 10021, USA.
Mol Cell Biol. 2010 Mar;30(5):1254-68. doi: 10.1128/MCB.00986-09. Epub 2009 Dec 14.
Rap1 (repressor activator protein 1) is a conserved multifunctional protein initially identified as a transcriptional regulator of ribosomal protein genes in Saccharomyces cerevisiae but subsequently shown to play diverse functions at multiple chromosomal loci, including telomeres. The function of Rap1 appears to be evolutionarily plastic, especially in the budding yeast lineages. We report here our biochemical and molecular genetic characterizations of Candida albicans Rap1, which exhibits an unusual, miniaturized domain organization in comparison to the S. cerevisiae homologue. We show that in contrast to S. cerevisiae, C. albicans RAP1 is not essential for cell viability but is critical for maintaining normal telomere length and structure. The rap1 null mutant exhibits drastic telomere-length dysregulation and accumulates high levels of telomere circles, which can be largely attributed to aberrant recombination activities at telomeres. Analysis of combination mutants indicates that Rap1 and other telomere proteins mediate overlapping but nonredundant roles in telomere protection. Consistent with the telomere phenotypes of the mutant, C. albicans Rap1 is localized to telomeres in vivo and recognizes the unusual telomere repeat unit with high affinity and sequence specificity in vitro. The DNA-binding Myb domain of C. albicans Rap1 is sufficient to suppress most of the telomere aberrations observed in the null mutant. Notably, we were unable to detect specific binding of C. albicans Rap1 to gene promoters in vivo or in vitro, suggesting that its functions are more circumscribed in this organism. Our findings provide insights on the evolution and mechanistic plasticity of a widely conserved and functionally critical telomere component.
Rap1(转录激活蛋白 1)是一种保守的多功能蛋白,最初在酿酒酵母中被鉴定为核糖体蛋白基因的转录调控因子,但随后在多个染色体位置(包括端粒)显示出多种功能。Rap1 的功能似乎具有进化可塑性,尤其是在出芽酵母谱系中。我们在这里报告了我们对白色念珠菌 Rap1 的生化和分子遗传特征的研究,与酿酒酵母同源物相比,它表现出不寻常的简化结构域组织。我们表明,与酿酒酵母不同,C. albicans RAP1 不是细胞活力所必需的,但对于维持正常的端粒长度和结构至关重要。rap1 缺失突变体表现出明显的端粒长度失调,并积累高水平的端粒环,这主要归因于端粒的异常重组活性。组合突变体的分析表明,Rap1 和其他端粒蛋白在端粒保护中发挥重叠但非冗余的作用。与突变体的端粒表型一致,C. albicans Rap1 在体内定位于端粒,并在体外以高亲和力和序列特异性识别不寻常的端粒重复单元。C. albicans Rap1 的 DNA 结合 Myb 结构域足以抑制在缺失突变体中观察到的大多数端粒异常。值得注意的是,我们无法在体内或体外检测到 C. albicans Rap1 与基因启动子的特异性结合,这表明其在该生物体中的功能更为有限。我们的发现为广泛保守且功能关键的端粒成分的进化和机制可塑性提供了新的见解。