Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto, Japan.
Department of Molecular Genetics, Cleveland Clinic Lerner College of Medicine, Cleveland, Ohio, United States of America.
PLoS Genet. 2019 Aug 27;15(8):e1008335. doi: 10.1371/journal.pgen.1008335. eCollection 2019 Aug.
Genomic rearrangements (gross chromosomal rearrangements, GCRs) threatens genome integrity and cause cell death or tumor formation. At the terminus of linear chromosomes, a telomere-binding protein complex, called shelterin, ensures chromosome stability by preventing chromosome end-to-end fusions and regulating telomere length homeostasis. As such, shelterin-mediated telomere functions play a pivotal role in suppressing GCR formation. However, it remains unclear whether the shelterin proteins play any direct role in inhibiting GCR at non-telomeric regions. Here, we have established a GCR assay for the first time in fission yeast and measured GCR rates in various mutants. We found that fission yeast cells lacking shelterin components Taz1 or Rap1 (mammalian TRF1/2 or RAP1 homologues, respectively) showed higher GCR rates compared to wild-type, accumulating large chromosome deletions. Genetic dissection of Rap1 revealed that Rap1 contributes to inhibiting GCRs via two independent pathways. The N-terminal BRCT-domain promotes faithful DSB repair, as determined by I-SceI-mediated DSB-induction experiments; moreover, association with Poz1 mediated by the central Poz1-binding domain regulates telomerase accessibility to DSBs, leading to suppression of de novo telomere additions. Our data highlight unappreciated functions of the shelterin components Taz1 and Rap1 in maintaining genome stability, specifically by preventing non-telomeric GCRs.
基因组重排（染色体重大结构重排，GCRs）威胁着基因组的完整性，并导致细胞死亡或肿瘤形成。在线性染色体的末端，一种称为 shelterin 的端粒结合蛋白复合物通过防止染色体端到端融合和调节端粒长度稳态来确保染色体的稳定性。因此，shelterin 介导的端粒功能在抑制 GCR 形成中起着关键作用。然而，目前尚不清楚 shelterin 蛋白是否在非端粒区域直接抑制 GCR 的形成。在这里，我们首次在裂殖酵母中建立了 GCR 测定法，并测量了各种突变体中的 GCR 率。我们发现，缺乏 shelterin 成分 Taz1 或 Rap1（分别为哺乳动物 TRF1/2 或 RAP1 同源物）的裂殖酵母细胞与野生型相比表现出更高的 GCR 率，积累了大量染色体缺失。Rap1 的遗传分析表明，Rap1 通过两种独立的途径抑制 GCR。N 端 BRCT 结构域通过 I-SceI 介导的 DSB 诱导实验促进了 DSB 的忠实修复；此外，通过中央 Poz1 结合结构域与 Poz1 的结合调节了端粒酶对 DSB 的可及性，从而抑制了从头添加端粒。我们的数据突出了 shelterin 成分 Taz1 和 Rap1 在维持基因组稳定性方面的未被认识的功能，特别是通过防止非端粒 GCR。
