Evolution of Telomeres in Schizosaccharomyces pombe and Its Possible Relationship to the Diversification of Telomere Binding Proteins

Telomeres of nuclear chromosomes are usually composed of an array of tandemly repeated sequences that are recognized by specific Myb domain containing DNA-binding proteins (telomere-binding proteins, TBPs). Whereas in many eukaryotes the length and sequence of the telomeric repeat is relatively conserved, telomeric sequences in various yeasts are highly variable. Schizosaccharomyces pombe provides an excellent model for investigation of co-evolution of telomeres and TBPs. First, telomeric repeats of S. pombe differ from the canonical mammalian type TTAGGG sequence. Second, S. pombe telomeres exhibit a high degree of intratelomeric heterogeneity. Third, S. pombe contains all types of known TBPs (Rap1p [a version unable to bind DNA], Tay1p/Teb1p, and Taz1p) that are employed by various yeast species to protect their telomeres. With the aim of reconstructing evolutionary paths leading to a separation of roles between Teb1p and Taz1p, we performed a comparative analysis of the DNA-binding properties of both proteins using combined qualitative and quantitative biochemical approaches. Visualization of DNA-protein complexes by electron microscopy revealed qualitative differences of binding of Teb1p and Taz1p to mammalian type and fission yeast telomeres. Fluorescence anisotropy analysis quantified the binding affinity of Teb1p and Taz1p to three different DNA substrates. Additionally, we carried out electrophoretic mobility shift assays using mammalian type telomeres and native substrates (telomeric repeats, histone-box sequences) as well as their mutated versions. We observed relative DNA sequence binding flexibility of Taz1p and higher binding stringency of Teb1p when both proteins were compared directly to each other. These properties may have driven replacement of Teb1p by Taz1p as the TBP in fission yeast.细胞核染色体的端粒通常由串联重复序列阵列构成，可被特定的含Myb结构域的DNA结合蛋白（端粒结合蛋白，telomere-binding proteins, TBPs）识别。尽管多数真核生物的端粒重复序列在长度与序列上相对保守，但各类酵母的端粒序列却呈现高度变异性。粟酒裂殖酵母（Schizosaccharomyces pombe）是研究端粒与端粒结合蛋白协同演化的极佳模型：其一，其端粒重复序列不同于经典的哺乳动物型TTAGGG序列；其二，该酵母的端粒展现出极高的端粒内部异质性；其三，粟酒裂殖酵母包含目前已知的所有类型端粒结合蛋白——Rap1p（一种无法结合DNA的亚型）、Tay1p/Teb1p与Taz1p，这些蛋白被多种酵母物种用于保护自身端粒。为了重构驱动Teb1p与Taz1p功能分化的演化路径，我们采用定性与定量生化联用方法，对两种蛋白的DNA结合特性开展了比较分析。通过电子显微镜可视化DNA-蛋白复合物，我们发现Teb1p与Taz1p结合哺乳动物型及裂殖酵母端粒的模式存在定性差异。荧光各向异性分析定量测定了Teb1p与Taz1p对三种不同DNA底物的结合亲和力。此外，我们还使用哺乳动物型端粒、天然底物（端粒重复序列、组蛋白框序列）及其突变体版本开展了电泳迁移率变动实验。直接对比两种蛋白的结合特性后，我们观察到Taz1p具有相对更强的DNA序列结合灵活性，而Teb1p的结合严格性更高。这些特性或许推动了粟酒裂殖酵母中Teb1p被Taz1p取代为端粒结合蛋白的演化进程。