Shelterin-like proteins and Yku inhibit nucleolytic processing of Saccharomyces cerevisiae telomeres.
Shelterin-like proteins and Yku inhibit nucleolytic processing of Saccharomyces cerevisiae telomeres.
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DOI：
10.1371/journal.pgen.1000966
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发表时间：
2010-05-27
期刊：
影响因子：
4.5
通讯作者：
Longhese MP
中科院分区：
文献类型：
--
作者：
Bonetti D;Clerici M;Anbalagan S;Martina M;Lucchini G;Longhese MP
真核细胞通过将它们包装成称为端粒的保护性结构来区分它们的染色体末端与意外的DNA双链断裂（DSB），端粒防止DNA修复/重组活动。在这里，我们调查的作用，保护芽殖酵母端粒降解的关键端粒蛋白。我们发现，酿酒酵母shelterin样蛋白Rif 1，Rif 2和Rap 1抑制核溶解过程中从头和天然端粒在G1和G2细胞周期阶段，与Rif 2和Rap 1表现出最强的效果。此外，Yku阻止端粒切除G1，独立于其在非同源末端连接的作用。Yku和shelterin样蛋白在抑制DNA降解在G1从头端粒，其中Yku起着主要的作用，防止启动，而Rif 1，Rif 2，Rap 1的作用主要是通过限制广泛的切除加合效应。事实上，从头端粒的核酸外切降解在yku 70 Δ中比在rif 2 Δ G1细胞中更有效，但在缺乏Yku的细胞中ssDNA的产生限于靠近端粒尖端的DNA区域。这种有限的加工是由于Rap 1，Rif 1和Rif 2的抑制作用，因为它们的失活不仅允许野生型而且也允许yku 70 Δ G1细胞中广泛的端粒切除。最后，Rap 1和Rif 2通过抑制MRX进入端粒来防止端粒降解，端粒也被Yku保护免受Exo 1核酸酶的影响。因此，染色体末端降解是由特异性抑制不同核酸酶作用的端粒蛋白控制的。端粒是一种特殊的核蛋白复合物，它将线性染色体的自然末端与染色体内双链断裂区分开来。事实上，端粒被保护免受DNA损伤检查点、同源重组或端对端融合的影响，这些检查点、同源重组或端对端融合通常促进染色体内DNA断裂的修复。当染色体末端保护失败时，功能失调的端粒被DNA修复和重组装置靶向，其结果从产生染色体异常（人类癌细胞的一般标志）到永久性细胞周期停滞和细胞死亡。虽然一些研究解决了端粒功能障碍的后果，但实现端粒保护的机制仍有待确定。在这里，我们调查这个问题，通过分析进化保守的端粒蛋白在保护芽殖酵母端粒降解的作用。我们证明，关键的端粒蛋白Yku，Rap 1，Rif 1和Rif 2抑制端粒降解，通过特异性地阻止不同的核酸酶的作用。由于这些蛋白质在芽殖酵母和哺乳动物细胞之间功能保守，它们也可能在防止人类端粒降解方面发挥关键作用。
Eukaryotic cells distinguish their chromosome ends from accidental DNA double-strand breaks (DSBs) by packaging them into protective structures called telomeres that prevent DNA repair/recombination activities. Here we investigate the role of key telomeric proteins in protecting budding yeast telomeres from degradation. We show that the Saccharomyces cerevisiae shelterin-like proteins Rif1, Rif2, and Rap1 inhibit nucleolytic processing at both de novo and native telomeres during G1 and G2 cell cycle phases, with Rif2 and Rap1 showing the strongest effects. Also Yku prevents telomere resection in G1, independently of its role in non-homologous end joining. Yku and the shelterin-like proteins have additive effects in inhibiting DNA degradation at G1 de novo telomeres, where Yku plays the major role in preventing initiation, whereas Rif1, Rif2, and Rap1 act primarily by limiting extensive resection. In fact, exonucleolytic degradation of a de novo telomere is more efficient in yku70Δ than in rif2Δ G1 cells, but generation of ssDNA in Yku-lacking cells is limited to DNA regions close to the telomere tip. This limited processing is due to the inhibitory action of Rap1, Rif1, and Rif2, as their inactivation allows extensive telomere resection not only in wild-type but also in yku70Δ G1 cells. Finally, Rap1 and Rif2 prevent telomere degradation by inhibiting MRX access to telomeres, which are also protected from the Exo1 nuclease by Yku. Thus, chromosome end degradation is controlled by telomeric proteins that specifically inhibit the action of different nucleases. Telomeres are specialized nucleoprotein complexes that distinguish the natural ends of linear chromosomes from intrachromosomal double-strand breaks. In fact, telomeres are protected from DNA damage checkpoints, homologous recombination, or end-to-end fusions that normally promote repair of intrachromosomal DNA breaks. When chromosome end protection fails, dysfunctional telomeres are targeted by the DNA repair and recombination apparatus, whose outcomes range from the generation of chromosomal abnormalities, general hallmarks for human cancer cells, to permanent cell cycle arrest and cell death. While several studies address the consequences of telomere dysfunctions, the mechanisms by which telomere protection is achieved remain to be determined. Here, we investigate this issue by analyzing the role of evolutionarily conserved telomeric proteins in protecting budding yeast telomeres from degradation. We demonstrate that the key telomeric proteins Yku, Rap1, Rif1, and Rif2 inhibit telomere degradation by specifically preventing the action of different nucleases. As these proteins are functionally conserved between budding yeast and mammalian cells, they might also play critical roles in preventing telomere degradation in humans.
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