Abstract
Here, we present a modified in vitro end-joining (EJ) assay to quantify EJ capacity, accuracy as well as pathway switch to alternative end-joining (Alt-EJ) or single strand annealing (SSA). A novel transformation assay was established to specifically measure circular repair products, which correlate with classical EJ efficiency. The EJ assay was validated using EJ-deficient mammalian cell lines (Ku80, DNA-PKcs, LigIV, or XRCC4 mutants). A pathway switch to Alt-EJ and SSA was seen exclusively in Ku-deficient cells. Circular EJ product formation correlated with cell survival and DSB repair capacity after X-irradiation. Investigation of 14 HNSCC cell lines revealed differences in the total EJ capacity but a broader variation in the amount of circular repair products. Sequencing of repair junctions in HNSCC cells demonstrated a predominance of high-fidelity EJ and an avoidance of both Alt-EJ and SSA. A significant correlation was observed between the amount of circular repair products, repair of IR-induced DSB and radiosensitivity. Collectively, these data indicate that the presented in vitro-EJ-assay can not only estimate the repair capacity of cancer cells to enable the stratification into radiosensitive or radioresistant, but can also identify repair pathway deregulation such as a switch to Alt-EJ or SSA, which enables tumor targeting.
Highlights
Ionizing radiation (IR) kills cells mainly by damaging DNA
The linearized plasmid was generated by digestion of pEJ-1200 plasmid using HindIII and PstI restriction enzymes, generating non-cohesive DNA-ends (Figure S1A), which will only be repaired by an active repair process
This is attributed to the fact that at 37◦C the nucleolytic activity within the cell free extracts (CFEs) exceeds the end joining activity and degradation of plasmids is faster than their repair [34]
Summary
Ionizing radiation (IR) kills cells mainly by damaging DNA. Among IR-induced damages, DNA double-strand breaks (DSBs) are considered to be the most critical lesion [1]. Most of the induced DSBs will be efficiently repaired, few will either be un- or mis-repaired, leading to lethal chromosomal aberrations and eventually cell death [2]. A strong correlation between DSB repair capacity and cell survival after IR was reported [3,4,5,6,7,8]. A minimal reduction in DSB repair capacity will profoundly impact the cellular radiosensitivity [9].
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