Radioresistance, DNA Damage and DNA Repair in Cells With Moderate Overexpression of RPA1
Radioresistance, DNA Damage and DNA Repair in Cells With Moderate Overexpression of RPA1
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DOI：
10.3389/fgene.2020.00855
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发表时间：
2020-07
影响因子：
3.7
通讯作者：
I. Velegzhaninov;E. Belykh;E. Rasova;Y. Pylina;D. Shadrin;D. Klokov
中科院分区：
文献类型：
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作者：
I. Velegzhaninov;E. Belykh;E. Rasova;Y. Pylina;D. Shadrin;D. Klokov
对遗传毒性应激（如电离辐射）的分子反应错综复杂，涉及数百个基因。是否有针对性地过表达内源性基因可以增强对电离辐射的抵抗力仍有待探索。在本研究中，我们利用CRISPR/dCas 9技术适度过表达编码复制蛋白A（RPA）关键功能亚基的RPA 1基因。RPA是一种高度保守的异源三聚体单链DNA结合蛋白复合物，参与DNA复制、重组和修复。RPA 1的功能障碍对细胞和生物体是有害的，并可能导致对许多应激因素的抵抗力下降。我们证明了过表达RPA 1的HEK 293 T细胞对γ-辐射的细胞杀伤表现出增强的抗性。使用碱彗星试验，我们显示了显着的加速DNA断裂后重新加入γ射线照射RPA 1过表达细胞。然而，自发的DNA损伤率也较高的RPA 1过表达的存在下，这表明在复制错误的处理，由于RPA蛋白的活性升高的改变。此外，对具有不同DNA损伤水平的细胞的分布的分析显示了RPA 1过表达与差异损伤的细胞亚群内的DNA修复动力学之间的联系。我们的研究结果提供了已知的DNA损伤应激反应的知识，并表明，通过靶向改变单个基因的表达来增强辐射抗性的概念是可行的，但应考虑和评估不良后果。
Molecular responses to genotoxic stress, such as ionizing radiation, are intricately complex and involve hundreds of genes. Whether targeted overexpression of an endogenous gene can enhance resistance to ionizing radiation remains to be explored. In the present study we take an advantage of the CRISPR/dCas9 technology to moderately overexpress the RPA1 gene that encodes a key functional subunit of the replication protein A (RPA). RPA is a highly conserved heterotrimeric single-stranded DNA-binding protein complex involved in DNA replication, recombination, and repair. Dysfunction of RPA1 is detrimental for cells and organisms and can lead to diminished resistance to many stress factors. We demonstrate that HEK293T cells overexpressing RPA1 exhibit enhanced resistance to cell killing by gamma-radiation. Using the alkali comet assay, we show a remarkable acceleration of DNA breaks rejoining after gamma-irradiation in RPA1 overexpressing cells. However, the spontaneous rate of DNA damage was also higher in the presence of RPA1 overexpression, suggesting alterations in the processing of replication errors due to elevated activity of the RPA protein. Additionally, the analysis of the distributions of cells with different levels of DNA damage showed a link between the RPA1 overexpression and the kinetics of DNA repair within differentially damaged cell subpopulations. Our results provide knew knowledge on DNA damage stress responses and indicate that the concept of enhancing radioresistance by targeted alteration of the expression of a single gene is feasible, however undesired consequences should be considered and evaluated.