INSERM U981, Gustave Roussy, 94805, Villejuif, France; Faculté de médecine, Université Paris-Sud, 94270, Kremlin-Bicêtre, France.
UF de Pharmacologie Biologie, Saint Louis Hopital, Institut de Génétique Moléculaire, 75010, Paris, France.
DNA Repair (Amst). 2018 Apr;64:34-44. doi: 10.1016/j.dnarep.2018.02.002. Epub 2018 Feb 11.
ERCC1/XPF endonuclease plays an important role in multiple DNA repair pathways and stands as a potential prognostic and predictive biomarker for cisplatin-based chemotherapy. Four distinct ERCC1 isoforms arising from alternative splicing have been described (201, 202, 203 and 204) but only the 202 isoform is functional in DNA excision repair, when interacting with its obligate partner XPF. Currently, there is no tool to assess specifically the expression of ERCC1-202 due to high sequence homology between the four isoforms. Here, we generated monoclonal antibodies directed against the heterodimer of ERCC1 and its obligate interacting partner XPF by genetic immunization. We obtained three monoclonal antibodies (2C11, 7C3 and 10D10) recognizing specifically the heterodimer ERCC1-202/XPF as well as the ERCC1-204/XPF with no affinity to ERCC1 or XPF monomers. By combining one of these three heterodimer-specific antibodies with a commercial anti-ERCC1 antibody (clone 4F9) unable to recognize the 204 isoform in a proximity ligation assay (PLA), we managed to specifically detect the functional ERCC1-202 isoform. This methodological breakthrough can constitute a basis for the development of clinical tests to evaluate ERCC1 functional proficiency.
核苷酸切除修复交叉互补基因 1/解旋酶 XPF（ERCC1/XPF）内切酶在多种 DNA 修复途径中发挥重要作用，是顺铂类化疗的潜在预后和预测生物标志物。已经描述了四种不同的通过选择性剪接产生的 ERCC1 异构体（201、202、203 和 204），但只有与其必需伴侣 XPF 相互作用的 202 异构体在 DNA 切除修复中具有功能。目前，由于四个异构体之间的高度序列同源性，没有专门评估 ERCC1-202 表达的工具。在这里，我们通过遗传免疫生成了针对 ERCC1 和其必需相互作用伴侣 XPF 异源二聚体的单克隆抗体。我们获得了三种单克隆抗体（2C11、7C3 和 10D10），它们特异性识别 ERCC1-202/XPF 异源二聚体以及 ERCC1-204/XPF，与 ERCC1 或 XPF 单体没有亲和力。通过将这三种异源二聚体特异性抗体之一与一种无法在邻近连接分析 (PLA) 中识别 204 异构体的商业抗 ERCC1 抗体（克隆 4F9）结合使用，我们成功地特异性检测到功能性 ERCC1-202 异构体。这一方法学上的突破可以为开发评估 ERCC1 功能能力的临床检测奠定基础。
