Radioresistance, DNA Damage and DNA Repair in Cells With Moderate Overexpression of RPA1

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 gammairradiation 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.

Actin nucleation safeguards single-stranded DNA and promotes replication fork protection

“…To efficiently support these processes, cells maintain a high level of RPA in the nucleus throughout the S-phase (around 8fold excess of what is required to support unperturbed replication 10 ). However, increased RPA levels can be toxic and drive genome instability and cancer progression, likely due to excessive formation of RPA-ssDNA complexes 10,14,[41][42][43] . Nonetheless, the prevailing view in the field is that the RPA molecules are freely diffused throughout the nucleus and bind rapidly to available ssDNA by a passive mechanism.…”

A Century of Fractionated Radiotherapy: How Mathematical Oncology Can Break the Rules

“…Repair: A number of studies investigated the importance of differences in DNA repair potential within radioresistant and sensitive cell populations [8,[39][40][41]. Pre-activation of pathways associated with DNA single and double strand break repair could here be linked to radioresistance.…”

DNA Damage Response and Cancer Metastasis: Clinical Implications and Therapeutic Opportunities

“…Several groups have highlighted a relationship between DNA repair and radioresistance. Upregulated DNA damage reaction was observed in multiple studies of cancer radioresistance (62)(63)(64) and was the intrinsic mechanisms associated with radioresistance of cancer stem cells (65). In a cellular model, acquired radioresistance was the result of alterations in DDR mediated by cyclin D1 overexpression, resulting in forced progression of S-phase and DDR activation (66).…”