BACKGROUND The C9ORF72 hexanucleotide repeat expansion is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two fatal age-related neurodegenerative diseases. The C9ORF72 expansion encodes five dipeptide repeat proteins (DPRs) that are produced through a non-canonical translation mechanism. Among the DPRs, proline-arginine (PR), glycine-arginine (GR), and glycine-alanine (GA) are the most neurotoxic and increase the frequency of DNA double strand breaks (DSBs). While the accumulation of these genotoxic lesions is increasingly recognized as a feature of disease, the mechanism(s) of DPR-mediated DNA damage are ill-defined and the effect of DPRs on the efficiency of each DNA DSB repair pathways has not been previously evaluated. METHODS AND RESULTS Using DNA DSB repair assays, we evaluated the efficiency of specific repair pathways, and found that PR, GR and GA decrease the efficiency of non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ), but not homologous recombination (HR). We found that PR inhibits DNA DSB repair, in part, by binding to the nucleolar protein nucleophosmin (NPM1). Depletion of NPM1 inhibited NHEJ and SSA, suggesting that NPM1 loss-of-function in PR expressing cells leads to impediments of both non-homologous and homology-directed DNA DSB repair pathways. By deleting NPM1 sub-cellular localization signals, we found that PR binds NPM1 regardless of the cellular compartment to which NPM1 was directed. Deletion of the NPM1 acidic loop motif, known to engage other arginine-rich proteins, abrogated PR and NPM1 binding. Using confocal and super-resolution immunofluorescence microscopy, we found that levels of RAD52, a component of the SSA repair machinery, were significantly increased iPSC neurons relative to isogenic controls in which the C9ORF72 expansion had been deleted using CRISPR/Cas9 genome editing. Western analysis of post-mortem brain tissues confirmed that RAD52 immunoreactivity is significantly increased in C9ALS/FTD samples as compared to controls. CONCLUSIONS Collectively, we characterized the inhibitory effects of DPRs on key DNA DSB repair pathways, identified NPM1 as a facilitator of DNA repair that is inhibited by PR, and revealed deficits in homology-directed DNA DSB repair pathways as a novel feature of C9ORF72-related disease.

中文翻译：

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二肽重复蛋白抑制C9ORF72 ALS / FTD中同源性指导的DNA双链断裂修复。

背景技术C9ORF72六核苷酸重复扩增是肌萎缩性侧索硬化症（ALS）和额颞痴呆（FTD）（两种致命的年龄相关性神经退行性疾病）的最常见已知遗传原因。C9ORF72扩展编码通过非规范翻译机制产生的五个二肽重复蛋白（DPR）。在DPR中，脯氨酸-精氨酸（PR），甘氨酸-精氨酸（GR）和甘氨酸-丙氨酸（GA）具有最大的神经毒性，并会增加DNA双链断裂（DSB）的频率。虽然这些遗传毒性损伤的积累越来越被认为是疾病的特征，但DPR介导的DNA损伤的机制尚不明确，并且DPR对每种DNA DSB修复途径效率的影响尚未得到评估。 。方法和结果使用DNA DSB修复测定法，我们评估了特定修复途径的效率，发现PR，GR和GA降低了非同源末端连接（NHEJ），单链退火（SSA）和微同源介导末端连接（MMEJ）的效率，但没有同源性重组（HR）。我们发现PR抑制DNA DSB修复的部分原因是通过结合至核仁蛋白核磷素（NPM1）。NPM1的耗竭抑制了NHEJ和SSA，这表明PR表达细胞中NPM1的功能丧失导致非同源和同源性导向的DNA DSB修复途径的障碍。通过删除NPM1亚细胞定位信号，我们发现PR绑定NPM1，而与NPM1所针对的细胞室无关。已知与其他富含精氨酸的蛋白质结合的NPM1酸性环基序的缺失，消除了PR和NPM1的结合。使用共聚焦和超高分辨率免疫荧光显微镜技术，我们发现RAD52（SSA修复机制的一个组成部分）的水平相对于使用CRISPR / Cas9基因组编辑已删除C9ORF72扩展的等基因对照显着增加了iPSC神经元。验尸后脑组织的Western分析证实，与对照组相比，C9ALS / FTD样品中RAD52免疫反应性显着提高。结论集体，我们表征了DPRs对关键DNA DSB修复途径的抑制作用，确定NPM1是PR抑制的DNA修复的促进剂，并揭示了同源性指导的DNA DSB修复途径的缺陷是C9ORF72相关的新特征。疾病。与等位基因对照相比，iPSC神经元的数量显着增加，其中等位基因对照已经使用CRISPR / Cas9基因组编辑删除了C9ORF72的扩增。验尸后脑组织的Western分析证实，与对照组相比，C9ALS / FTD样品中RAD52免疫反应性显着提高。结论集体，我们表征了DPRs对关键DNA DSB修复途径的抑制作用，确定NPM1是PR抑制的DNA修复的促进剂，并揭示了同源性指导的DNA DSB修复途径的缺陷是C9ORF72相关的新特征。疾病。与等位基因对照相比，iPSC神经元的数量显着增加，其中等位基因对照已经使用CRISPR / Cas9基因组编辑删除了C9ORF72的扩增。验尸后脑组织的Western分析证实，与对照组相比，C9ALS / FTD样品中RAD52免疫反应性显着提高。结论集体，我们表征了DPRs对关键DNA DSB修复途径的抑制作用，确定NPM1是PR抑制的DNA修复的促进剂，并揭示了同源性指导的DNA DSB修复途径的缺陷是C9ORF72相关的新特征。疾病。验尸后脑组织的Western分析证实，与对照组相比，C9ALS / FTD样品中RAD52免疫反应性显着提高。结论集体，我们表征了DPRs对关键DNA DSB修复途径的抑制作用，确定NPM1是PR抑制的DNA修复的促进剂，并揭示了同源性指导的DNA DSB修复途径的缺陷是C9ORF72相关的新特征。疾病。验尸后脑组织的Western分析证实，与对照组相比，C9ALS / FTD样品中RAD52免疫反应性显着提高。结论集体，我们表征了DPRs对关键DNA DSB修复途径的抑制作用，确定NPM1是PR抑制的DNA修复的促进剂，并揭示了同源性指导的DNA DSB修复途径的缺陷是C9ORF72相关的新特征。疾病。