Surveillance and maintenance of DNA ends

描述（由申请人提供）：端粒是线性真核生物染色体末端的核蛋白复合物，与DNA双链断裂（DSBs）不同。为了保持基因组的完整性，所有生物体对dsb的反应都是迅速启动dna损伤反应。这种反应涉及DNA修复因子的招募到DNA损伤位点和信号转导途径的激活，通常称为DNA损伤检查点途径。端粒从检查点激活和DNA修复中逃脱。矛盾的是，检查点蛋白和修复蛋白对端粒的维持至关重要。该项目的长期目标是揭示端粒如何与DSB反应机制合作的调控机制。dsb通过同源重组（HR）或非同源末端连接（NHEJ）修复。Mre11-Rad50-Nbs1（出芽酵母中的Xrs2）复合体识别DSB并促进HR和NHEJ修复，而Ku复合体结合DSB末端并刺激NHEJ途径。ATM激酶在酵母和人类中高度保守，在DSB诱导下激活检查点通路中起关键作用。ATM激酶在出芽酵母中由TEL1编码。ATM/Tel1通过与Mre11-Rad50-Nbs1/Xrs2 （MRN/MRX）复合物相互作用定位到dsb。端粒蛋白如何调节ATM/Tel1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着端粒特异性DNA结合蛋白，包括TRF1和TRF2。TRF2与端粒相互作用并将RAP1招募到端粒。人类TRF2-RAP1复合体抑制ATM检查点和NHEJ通路，尽管分子细节尚不完全清楚。在出芽酵母中，Rap1 （Rap1同源物）直接与双链端粒序列结合，在端粒稳态中起关键作用。出芽酵母Rap1将包括Rif1在内的几种端粒结合蛋白招募到端粒。在本提案中，我们计划揭示Rif1如何控制DNA末端的Tel1积累和DSB修复的分子细节（Aim 1），并定义Rap1如何抑制DNA末端MRX积累的机制（Aim 2）。我们还将确定当端粒抑制NHEJ通路时Ku如何定位于端粒（目的3）。鉴于DNA修复和端粒结合蛋白的进化守恒，我们使用出芽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。由于端粒维护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. PUBLIC HEALTH RELEVANCE: It is crucial to understand how telomeres modulate the DNA double-stranded break (DSB) response, because improper telomere maintenance leads to cancer development and cell senescence. Since DNA repair and telomere-binding proteins are conserved from yeast to human, our study using budding yeast will show the right direction to understand the picture of how human telomere-binding proteins regulate DSB repair and checkpoint response.