Understanding DNA break repair pathway choice regulation by the cNHEJ inhibitor CYREN

了解 cNHEJ 抑制剂 CYREN 的 DNA 断裂修复途径选择调节

• 批准号: 10397557
• 负责人: Jan Karlseder
• 金额: $ 43.13万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-06 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10397557
• 关键词: Affect; Alleles; Alternative Splicing; Amino Acids; Applications Grants; Binding; Cell Cycle; Cell Cycle Deregulation; Cell Cycle Regulation; Cell Cycle Stage; Cell Survival; Cell physiology; Cells; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; Dominant-Negative Mutation; Double Strand Break Repair; Ensure; Essential Amino Acids; Event; Excision; Exposure to; G2 Phase; G22P1 gene; Generations; Genome; Genome Stability; Genomic Instability; Genotoxic Stress; Goals; Human; Human Genome; Infection; Invaded; Lead; Learning; Lesion; Lysine; Maintenance; Malignant Neoplasms; Maps; Metabolism; Modeling; Modification; Mutagens; Mutate; Nature; Neoplastic Cell Transformation; Nonhomologous DNA End Joining; Open Reading Frames; Pathway interactions; Pharmaceutical Preparations; Play; Point Mutation; Predisposition; Proteasome Inhibition; Protein Isoforms; Proteins; Regulation; Role; S phase; Sister Chromatid; Site; System; Therapeutic Intervention; Translating; XRCC5 gene; base; brca gene; cancer cell; design; experimental study; genetic information; genome integrity; genotoxicity; homologous recombination; inhibitor; mutant; novel; overexpression; p53-binding protein 1; polypeptide; preservation; prevent; public health relevance; repaired; telomere

Project Summary DNA double stranded breaks (DSBs) are deleterious lesions that require rapid repair to avoid the loss of genetic information, genomic instability, neoplastic transformation and cancer formation. In human systems, the two predominant pathways for double stranded DNA break repair are canonical non-homologous end joining (cNHEJ) and homologous recombination (HR). The cNHEJ machinery recognizes breaks, indiscriminately joins them independent of sequence context and is therefore considered error prone and potentially genotoxic. HR relies on resection of the 5’ strand with the generation of single stranded 3’ overhangs, which invade homologous sister chromatids to promote error-free break repair. Choice between HR and cNHEJ depends primarily on the cell cycle stage and the nature of the break. During G1 of the cell cycle HR is inhibited by RIF1 and 53BP1, which prevent the required BRCA1/2 complex assembly and end resection for HR initiation. During S and G2, when sister chromatids are available as a template for HR, both cNHEJ and HR pathways can be employed and compete to repair DSBs. End resection is activated by CtIP in S and G2 phases and promotes HR, but it is unclear how the abundant and efficient cNHEJ machinery is suppressed in S and G2 to allow resection at break sites and commencement of HR, thereby ensuring error- free repair of lesions to preserve genome integrity. CYREN (Cell cYcle REgulator of NHEJ) was originally identified in a screen for potential modulators of retroviral infection. Later, an alternatively spliced isoform of CYREN, CYREN-2, was found as short open reading frame translated polypeptide and shown to interact with the Ku70/80 heterodimer, pointing at a potential role in cNHEJ. Here it is proposed to investigate the discovery that CYREN modulates the cell cycle dynamics of cNHEJ and that the small protein is a direct cell cycle regulator of cNHEJ. In three specific aims it is proposed to investigate the mechanism of how CYREN inhibits cNHEJ through the CYREN interaction with the Ku heterodimer complex (AIM 1), how CYREN is cell cycle regulated and controls the cell cycle regulation of DSB repair pathway choice (AIM2) and finally what the effects of CYREN deletion and overexpression are, how the deregulation of cell cycle control of cNHEJ influences genome maintenance and genome instability and whether cells that lack CYREN are sensitive to DNA damage causing agents (AIM 3). In summary, this grant proposal focuses on CYREN, a novel regulator of DNA repair pathway choice, the mechanism of how CYREN is regulated and controls cNHEJ, whether lack of CYREN causes genome instability and whether CYREN targeting can be exploited to sensitize cancer cells to treatment with genotoxic agents.

项目摘要 DNA双链断裂（DSB）是有害的损伤，其需要快速修复以避免DNA双链断裂的损失。 遗传信息、基因组不稳定性、肿瘤转化和癌症形成。在人类系统中， 双链DNA断裂修复两个主要途径是典型的非同源末端 连接（cNHEJ）和同源重组（HR）。cNHEJ机器识别断裂， 不加区别地连接它们而不依赖于序列上下文，因此被认为是容易出错的， 潜在的遗传毒性。HR依赖于5'链的切除，产生单链3'链。 突出端，其侵入同源姐妹染色单体以促进无错误的断裂修复。之间选择 HR和cNHEJ主要取决于细胞周期阶段和断裂的性质。在细胞的G1期 RIF 1和53 BP 1抑制周期HR，阻止所需的BRCA 1/2复合体组装并终止 切除术用于HR启动。在S期和G2期，当姐妹染色单体可作为HR的模板时， cNHEJ和HR途径可以被采用并竞争修复DSB。末端切除术由CtIP激活， S和G2阶段并促进HR，但尚不清楚丰富和有效的cNHEJ机制是如何 在S和G2中抑制，以允许在断裂部位切除和HR开始，从而确保错误- 自由修复损伤以保持基因组完整性。CYREN（NHEJ的细胞周期调节剂）最初是 在筛选逆转录病毒感染的潜在调节剂中鉴定。后来，一种选择性剪接的 CYREN，CYREN-2，被发现是短的开放阅读框翻译的多肽，并显示与 Ku 70/80异二聚体，指出在cNHEJ中的潜在作用。在这里，建议调查的发现， CYREN调节cNHEJ的细胞周期动力学，小蛋白是直接的细胞周期 cNHEJ的调节剂。在三个具体的目标，它提出了研究机制，如何CYREN抑制 cNHEJ通过CYREN与Ku异二聚体复合物（AIM 1）的相互作用，CYREN如何在细胞周期中起作用 调节和控制DSB修复途径选择（AIM 2）的细胞周期调节， CYREN缺失和过表达的影响是，cNHEJ细胞周期控制的失调如何影响cNHEJ细胞的生长， 影响基因组维持和基因组不稳定性，以及缺乏CYREN的细胞是否对 DNA损伤因子（AIM 3）。总之，这项拨款建议的重点是CYREN，一个新的监管机构 DNA修复途径的选择，CYREN如何调节和控制cNHEJ的机制，是否缺乏 CYREN引起基因组不稳定性，以及CYREN靶向是否可用于敏化癌细胞 遗传毒性药物治疗