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

项目摘要 DNA双链断裂(DSB)是一种有害的损伤，需要快速修复以避免 遗传信息、基因组不稳定、肿瘤转化和癌症形成。在人类系统中， 双链DNA断裂修复的两条主要途径是典型的非同源末端 连接(CNHEJ)和同源重组(HR)。CNHEJ的机器可以识别破损， 不分青红皂白地与序列上下文无关地联接它们，因此被认为容易出错 具有潜在的遗传毒性。HR依赖于5‘链切除和单链3’链的产生 突起，它侵入同源姐妹染色单体，促进无错误的断裂修复。在以下选项中选择 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 周期HR被Rif1和53BP1抑制，这阻止了所需的BRCA1/2复合体的组装和结束 切除以启动HR。在S和G2期间，当姐妹染色单体可用作HR的模板时，两者 CNHEJ和HR通路可用于修复DSB。末端切除由中的CtIP激活 S和G2分阶段提拔人力资源，但尚不清楚cNHEJ机制的充裕和高效程度 在S和G2中被抑制，以允许在断裂处切除和开始HR，从而确保错误- 免费修复病变以保持基因组的完整性。CyREN(NHEJ的细胞周期调节因子)最初是 在逆转录病毒感染的潜在调节剂的筛查中被确定。后来，另一种剪接的亚型 研究发现，CYREN-2是一种短开放阅读框翻译的多肽，并与其相互作用 Ku70/80异源二聚体，表明在cNHEJ中有潜在的作用。在这里，建议对这一发现进行调查 CyREN调控cNHEJ的细胞周期动力学，小蛋白是直接的细胞周期 CNHEJ的监管者。在三个特定的目标中，提出了研究CyREN如何抑制的机制 CNHEJ通过CyREN与Ku异源二聚体复合体(AIM 1)的相互作用，CyREN是如何影响细胞周期的 调控细胞周期调控的DSB修复途径选择(AIM2) CyREN缺失和过表达的影响是如何解除cNHEJ细胞周期调控的 影响基因组的维持和基因组的不稳定性，以及缺乏CYREN的细胞是否对 DNA损伤诱导剂(AIM 3)。综上所述，这项拨款提案聚焦于一种新的监管机构--CYREN 关于DNA修复途径的选择，CyREN是如何调控和控制cNHEJ的，是否缺乏 CyREN的缺失导致基因组不稳定以及是否可以利用CyREN靶向来使癌细胞增敏 用基因毒剂进行治疗。

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.