Abstract
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.
Highlights
Replication protein A (RPA) is a single-stranded DNA-binding protein complex that plays a significant role in maintaining the genome integrity by facilitating DNA replication, recombination, and repair (Audry et al, 2015)
DNA-binding domains (DBD)-A, -B, -C, and -F are located on the RPA1 subunit, whereas DBD-D and DBD-E are on RPA2 and RPA3, respectively (Maréchal and Zou, 2015)
We first examined the effectiveness of the CRISPRa/dCas9VPR system by measuring the RPA1 mRNA levels in cells transfected with dCas9-VPR and RPA1 sgRNAs
Summary
Replication protein A (RPA) is a single-stranded DNA-binding protein complex that plays a significant role in maintaining the genome integrity by facilitating DNA replication, recombination, and repair (Audry et al, 2015). DBD-F has a crucial role in DNA damage signaling Mutations in this part of the protein affecting the conformation disrupted the G2/M checkpoint, inhibited Ddc2/ATRIP and Ddc1/RAD9 interactions and prevented their recruitment to damage sites (Maréchal and Zou, 2015). Mutations of the DBD-A and DBDB domains of RPA1 lead to a disruption of DNA repair, but had no impact on replication (Hass et al, 2012). The physical interaction of these two proteins was necessary to protect the replication fork Consistent with this notion, a heterozygous mutation of RPA1 promoted tumorigenesis in mice (Wang et al, 2005, 2015). Cells with transient overexpression were treated with acute gamma-radiation and the survival rate and the rate of DNA repair were analyzed
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