Surveillance and maintenance of DNA ends

描述（由申请人提供）：端粒是位于线性真核染色体末端的核蛋白复合物，与DNA双链断裂（DSB）不同。为了维持基因组的完整性，所有生物体都会通过迅速启动DNA损伤反应来对DSB做出反应。这种反应涉及DNA修复因子到DNA损伤位点的募集和信号转导途径的激活，通常称为DNA损伤检查点途径。端粒逃避检查点激活和DNA修复。巧合的是，检查点和修复蛋白对于端粒的维持是必不可少的。 该项目的长期目标是揭示端粒如何与DSB反应机制合作的调节机制。DSB通过同源重组（HR）或非同源末端连接（NHEJ）修复。Mre 11-Rad 50-Nbs 1（出芽酵母中的Xrs 2）复合物识别DSB并促进HR和NHEJ修复，而Ku复合物结合DSB末端并刺激NHEJ途径。ATM激酶，这是高度保守的从酵母到人类，在激活检查点途径中起着关键作用，以响应DSB诱导。ATM激酶由出芽酵母中的TEL 1编码。ATM/Tel 1通过与Mre 11-Rad 50-Nbs 1/Xrs 2（MRN/MRX）复合物相互作用定位于DSB。端粒蛋白如何调节ATM/Tel 1检查点和Ku介导的NHEJ通路仍有待确定。端粒的双链部分覆盖有端粒特异性DNA结合蛋白，包括TRF 1和TRF 2。TRF 2与RAP 1相互作用并将RAP 1招募到端粒。人TRF 2-RAP 1复合物抑制ATM检查点和NHEJ通路，尽管分子细节尚未完全了解。在芽殖酵母中，Rap 1（RAP 1同源物）直接与双链端粒序列结合，并在端粒稳态中发挥关键作用。芽殖酵母Rap 1招募包括Rif 1在内的几种端粒结合蛋白到端粒。 在这项提案中，我们计划揭示Rif 1如何控制Tel 1积累和DNA末端DSB修复的分子细节（Aim 1），并定义Rap 1如何抑制DNA末端MRX积累的机制（Aim 2）。我们还将确定Ku如何定位于端粒，而端粒抑制NHEJ通路（目的3）。鉴于DNA修复和端粒结合蛋白的进化保守性，我们使用芽殖酵母的研究将为了解人类端粒如何调节DSB反应提供宝贵的信息。由于端粒维持不当与癌症发生和细胞衰老有关，我们的研究将有助于开发更好的癌症治疗和预防过早衰老。

DESCRIPTION (provided by applicant): Telomeres are nucleoprotein complexes at the ends of linear eukaryotic chromosomes, which are distinguished from DNA double-strand breaks (DSBs). To maintain genomic integrity, all organisms respond to DSBs by promptly launching the DNA-damage response. This response involves the recruitment of DNA repair factors to sites of DNA damage and the activation of signal transduction pathways, often termed DNA-damage checkpoint pathways. Telomeres escape from checkpoint activation and DNA repair. Paradoxically, checkpoint and repair proteins are essential for telomere maintenance. The long-term goal of this project is to uncover the regulatory mechanism of how telomeres collaborate with DSB response machinery. DSBs are repaired by homologous recombination (HR) or non-homologous end joining (NHEJ). The Mre11-Rad50-Nbs1 (Xrs2 in budding yeast) complex recognizes DSBs and promotes HR and NHEJ repair, whereas the Ku complex binds to DSB ends and stimulates the NHEJ pathway. The ATM kinase, which is highly conserved from yeast to human, plays a key role in activating the checkpoint pathway in response to DSB induction. ATM kinase is encoded by TEL1 in budding yeast. ATM/Tel1 localizes to DSBs by interacting Mre11-Rad50-Nbs1/Xrs2 (MRN/MRX) complex. How telomere proteins modulate the ATM/Tel1 checkpoint and Ku-mediated NHEJ pathway remains to be determined. Double-stranded portions of telomeres are covered with telomere-specific DNA binding proteins including TRF1 and TRF2. TRF2 interacts with and recruits RAP1 to telomeres. Human TRF2-RAP1 complex inhibits the ATM checkpoint and NHEJ pathways, although the molecular detail is not fully understood. In budding yeast, Rap1 (the RAP1 homolog) binds directly to double-stranded telomere sequences and plays a key role in telomere homeostasis. Budding yeast Rap1 recruits several telomere-binding proteins including Rif1 to telomeres. In this proposal, we plan to uncover the molecular detail of how Rif1 controls Tel1 accumulation and DSB repair at DNA ends (Aim 1), and define the mechanism of how Rap1 inhibits MRX accumulation at DNA ends (Aim 2). We will also determine how Ku localizes to telomeres while telomeres inhibit the NHEJ pathway (Aim 3). Given the evolutionary conservation of DNA repair and telomere-binding proteins, our study using budding yeast will provide invaluable information to understand how human telomeres modulate DSB responses. Since improper telomere maintenance is implicated in carcinogenesis and cell senescence, our study will contribute to the development of better cancer treatment and the prevention of premature aging.