Surveillance and maintenance of DNA ends

描述(申请人提供)：端粒是位于真核生物线性染色体末端的核蛋白复合体，区别于DNA双链断裂(DSB)。为了保持基因组的完整性，所有生物体都会迅速启动DNA损伤反应，从而对双链断裂做出反应。这种反应包括DNA修复因子在DNA损伤部位的募集和信号转导通路的激活，通常被称为DNA损伤检查点通路。端粒从检查点激活和DNA修复中逃脱。矛盾的是，检查点和修复蛋白是端粒维持所必需的。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。双链断裂可通过同源重组(HR)或非同源末端连接(NHEJ)修复。Mre11-Rad50-Nbs1(发芽酵母中的Xrs2)复合体识别DSB并促进HR和NHEJ修复，而Ku复合体与DSB末端结合并刺激NHEJ途径。从酵母到人类都高度保守的ATM激酶，在激活DSB诱导的检查点通路中起着关键作用。ATM激酶是由TEL1在芽殖酵母中编码的。ATM/Tel1通过与Mre11-Rad50-Nbs1/Xrs2(MRN/MRX)复合体相互作用而定位于DSB。端粒蛋白如何调节ATM/Tel1检查点和Ku介导的NHEJ途径仍有待确定。端粒的双链部分覆盖着端粒特异的DNA结合蛋白，包括TRF1和TRF2。TRF2与端粒相互作用，并招募RAP1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。在发芽酵母中，RAP1(RAP1同源物)直接与双链端粒序列结合，在端粒动态平衡中发挥关键作用。萌芽酵母Rap1招募了几种端粒结合蛋白，包括Rif1到端粒。在这项提议中，我们计划揭示Rif1如何控制Tel1积累和DNA末端DSB修复的分子细节(目标1)，并定义Rap1如何抑制DNA末端MRX积累的机制(目标2)。我们还将确定Ku如何定位于端粒，而端粒则抑制NHEJ途径(目标3)。鉴于DNA修复和端粒结合蛋白在进化上的保守性，我们使用发芽酵母的研究将为理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肿瘤的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方法和防止过早衰老。

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.