Melo-Hanchuk Talita Diniz, Slepicka Priscila Ferreira, Pelegrini Alessandra Luiza, Menck Carlos Frederico Martins, Kobarg Jörg
Departamento de Bioquímica e Biologia Tecidual, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil.
Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Campinas, São Paulo, Brazil.
J Cell Biochem. 2019 Oct;120(10):16853-16866. doi: 10.1002/jcb.28943. Epub 2019 May 15.
Cells are daily submitted to high levels of DNA lesions that trigger complex pathways and cellular responses by cell cycle arrest, apoptosis, alterations in transcriptional response, and the onset of DNA repair. Members of the NIMA-related kinase (NEK) family have been related to DNA damage response and repair and the first insight about NEK5 in this context is related to its role in centrosome separation resulting in defects in chromosome integrity. Here we investigate the potential correlation between NEK5 and the DNA damage repair index. The effect of NEK5 in double-strand breaks caused by etoposide was accessed by alkaline comet assay and revealed that NEK5-silenced cells are more sensitive to etoposide treatment. Topoisomerase IIβ (TOPIIβ) is a target of etoposide that leads to the production of DNA breaks. We demonstrate that NEK5 interacts with TOPIIβ, and the dynamics of this interaction is evaluated by proximity ligation assay. The complex NEK5/TOPIIβ is formed immediately after etoposide treatment. Taken together, the results of our study reveal that NEK5 depletion increases DNA damage and impairs proper DNA damage response, pointing out NEK5 as a potential kinase contributor to genomic stability.
细胞每天都会受到高水平的DNA损伤，这些损伤会通过细胞周期停滞、凋亡、转录反应改变以及DNA修复的启动来触发复杂的信号通路和细胞反应。NIMA相关激酶（NEK）家族成员与DNA损伤反应和修复有关，在此背景下，对NEK5的初步认识与其在中心体分离中的作用有关，进而导致染色体完整性缺陷。在这里，我们研究了NEK5与DNA损伤修复指数之间的潜在相关性。通过碱性彗星试验评估了NEK5对依托泊苷引起的双链断裂的影响，结果显示，沉默NEK5的细胞对依托泊苷治疗更为敏感。拓扑异构酶IIβ（TOPIIβ）是依托泊苷的作用靶点，会导致DNA断裂。我们证明NEK5与TOPIIβ相互作用，并通过邻近连接试验评估这种相互作用的动态变化。依托泊苷处理后立即形成NEK5/TOPIIβ复合物。综上所述，我们的研究结果表明，NEK5的缺失会增加DNA损伤并损害正常的DNA损伤反应，指出NEK5是基因组稳定性的潜在激酶贡献者。
