Lehmann Janin, Schubert Steffen, Seebode Christina, Apel Antje, Ohlenbusch Andreas, Emmert Steffen
Clinic and Policlinic for Dermatology and Venereology, University Medical Center Rostock, Rostock, Germany.
Information Network of Departments of Dermatology (IVDK), University Medical Center Goettingen, Goettingen, Germany.
Oncotarget. 2017 Dec 8;9(1):1012-1027. doi: 10.18632/oncotarget.23105. eCollection 2018 Jan 2.
The two endonucleases XPF and XPG are essentially involved in nucleotide excision repair (NER) and interstrand crosslink (ICL) repair. Defects in these two proteins result in severe diseases like xeroderma pigmentosum (XP). We applied our newly CRISPR/Cas9 generated human knockout cell line with complete loss of XPF and primary fibroblasts from an XP-G patient (XP20BE) to analyze until now uncharacterized spontaneous mRNA splice variants of these two endonucleases. Functional analyses of these variants were performed using luciferase-based reporter gene assays. Two and splice variants with residual repair capabilities in NER, as well as ICL repair could be identified. Almost all variants are severely C-terminally truncated and lack important protein-protein interaction domains. Interestingly, XPF-202, differing to XPF-003 in the first 12 amino acids only, had no repair capability at all, suggesting an important role of this region during DNA repair, potentially concerning protein-protein interaction. We also identified splice variants of and exerting inhibitory effects on NER. Moreover, we showed that the XPF and XPG splice variants presented with different inter-individual expression patterns in healthy donors, as well as in various tissues. With regard to their residual repair capability and dominant-negative effects, functionally relevant spontaneous and splice variants present promising prognostic marker candidates for individual cancer risk, disease outcome, or therapeutic success. This merits further investigations, large association studies, and translational research within clinical trials in the future.
两种核酸内切酶XPF和XPG主要参与核苷酸切除修复（NER）和链间交联（ICL）修复。这两种蛋白质的缺陷会导致严重疾病，如着色性干皮病（XP）。我们应用新的CRISPR/Cas9技术构建的完全缺失XPF的人类基因敲除细胞系以及来自一名XP-G患者（XP20BE）的原代成纤维细胞，来分析这两种核酸内切酶目前尚未被表征的自发mRNA剪接变体。使用基于荧光素酶的报告基因检测对这些变体进行功能分析。可以鉴定出两种在NER以及ICL修复中具有残余修复能力的剪接变体。几乎所有变体在C末端都严重截短，并且缺乏重要的蛋白质-蛋白质相互作用结构域。有趣的是，仅在前12个氨基酸上与XPF-003不同的XPF-202完全没有修复能力，这表明该区域在DNA修复过程中具有重要作用，可能与蛋白质-蛋白质相互作用有关。我们还鉴定出了对NER有抑制作用的XPG剪接变体。此外，我们发现XPF和XPG剪接变体在健康供体以及各种组织中呈现出不同的个体间表达模式。鉴于它们的残余修复能力和显性负效应，功能相关的自发XPF和XPG剪接变体有望成为个体癌症风险、疾病预后或治疗成功的有前景的预后标志物候选物。这值得在未来进行进一步研究、大规模关联研究以及临床试验中的转化研究。
