Radioresistance, DNA Damage and DNA Repair in Cells With Moderate Overexpression of RPA1
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 CRIS...
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| Format: | Article |
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Frontiers Media S.A.
2020-07-01
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| Series: | Frontiers in Genetics |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fgene.2020.00855/full |
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| author | Ilya O. Velegzhaninov Elena S. Belykh Elena E. Rasova Yana I. Pylina Dmitry M. Shadrin Dmitry Yu. Klokov Dmitry Yu. Klokov |
| author_facet | Ilya O. Velegzhaninov Elena S. Belykh Elena E. Rasova Yana I. Pylina Dmitry M. Shadrin Dmitry Yu. Klokov Dmitry Yu. Klokov |
| author_sort | Ilya O. Velegzhaninov |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-14T02:02:23Z |
| format | Article |
| id | doaj.art-208ea01d84124112ba46226f19fc6ebc |
| institution | Directory Open Access Journal |
| issn | 1664-8021 |
| language | English |
| last_indexed | 2024-12-14T02:02:23Z |
| publishDate | 2020-07-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Genetics |
| spelling | doaj.art-208ea01d84124112ba46226f19fc6ebc2022-12-21T23:20:58ZengFrontiers Media S.A.Frontiers in Genetics1664-80212020-07-011110.3389/fgene.2020.00855551292Radioresistance, DNA Damage and DNA Repair in Cells With Moderate Overexpression of RPA1Ilya O. Velegzhaninov0Elena S. Belykh1Elena E. Rasova2Yana I. Pylina3Dmitry M. Shadrin4Dmitry Yu. Klokov5Dmitry Yu. Klokov6Institute of Biology of Komi Scientific Centre of the Ural Branch of the Russian Academy of Sciences, Syktyvkar, RussiaInstitute of Biology of Komi Scientific Centre of the Ural Branch of the Russian Academy of Sciences, Syktyvkar, RussiaInstitute of Biology of Komi Scientific Centre of the Ural Branch of the Russian Academy of Sciences, Syktyvkar, RussiaInstitute of Biology of Komi Scientific Centre of the Ural Branch of the Russian Academy of Sciences, Syktyvkar, RussiaInstitute of Biology of Komi Scientific Centre of the Ural Branch of the Russian Academy of Sciences, Syktyvkar, RussiaInstitut de Radioprotection et de Sureté Nucléaire, PSE-SANTE, SESANE, LRTOX, Fontenay-aux-Roses, FranceDepartment of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON, CanadaMolecular 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.https://www.frontiersin.org/article/10.3389/fgene.2020.00855/fullRPA1 overexpressionCRISPRaradioresistanceDNA damageDNA repair |
| spellingShingle | Ilya O. Velegzhaninov Elena S. Belykh Elena E. Rasova Yana I. Pylina Dmitry M. Shadrin Dmitry Yu. Klokov Dmitry Yu. Klokov Radioresistance, DNA Damage and DNA Repair in Cells With Moderate Overexpression of RPA1 Frontiers in Genetics RPA1 overexpression CRISPRa radioresistance DNA damage DNA repair |
| title | Radioresistance, DNA Damage and DNA Repair in Cells With Moderate Overexpression of RPA1 |
| title_full | Radioresistance, DNA Damage and DNA Repair in Cells With Moderate Overexpression of RPA1 |
| title_fullStr | Radioresistance, DNA Damage and DNA Repair in Cells With Moderate Overexpression of RPA1 |
| title_full_unstemmed | Radioresistance, DNA Damage and DNA Repair in Cells With Moderate Overexpression of RPA1 |
| title_short | Radioresistance, DNA Damage and DNA Repair in Cells With Moderate Overexpression of RPA1 |
| title_sort | radioresistance dna damage and dna repair in cells with moderate overexpression of rpa1 |
| topic | RPA1 overexpression CRISPRa radioresistance DNA damage DNA repair |
| url | https://www.frontiersin.org/article/10.3389/fgene.2020.00855/full |
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