Abstract
This study investigated the efficiency of Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair systems in rejoining DNA double-strand breaks (DSB) induced in CCD-34Lu cells by different γ-ray doses. The kinetics of DNA repair was assessed by analyzing the fluorescence decrease of γ-H2AX foci measured by SOID (Sum Of Integrated Density) parameter and counting foci number in the time-interval 0.5–24 hours after irradiation. Comparison of the two methods showed that the SOID parameter was useful in determining the amount and the persistence of DNA damage signal after exposure to high or low doses of ionizing radiation. The efficiency of DSB rejoining during the cell cycle was assessed by distinguishing G1, S, and G2 phase cells on the basis of nuclear fluorescence of the CENP-F protein. Six hours after irradiation, γ-H2AX foci resolution was higher in G2 compared to G1 cells in which both NHEJ and HR can cooperate. The rejoining of γ-H2AX foci in G2 phase cells was, moreover, decreased by RI-1, the chemical inhibitor of HR, demonstrating that homologous recombination is at work early after irradiation. The relevance of HR in DSB repair was assessed in DNA-PK-deficient M059J cells and in CCD-34Lu treated with the DNA-PKcs inhibitor, NU7026. In both conditions, the kinetics of γ-H2AX demonstrated that DSBs repair was markedly affected when NHEJ was absent or impaired, even in G2 phase cells in which HR should be at work. The recruitment of RAD51 at DSB sites was, moreover, delayed in M059J and in NU7026 treated-CCD-34Lu, with respect to DNA-PKcs proficient cells and continued for 24 hours despite the decrease in DNA repair. The impairment of NHEJ affected the efficiency of the HR system and significantly decreased cell survival after ionizing radiation, confirming that DSB rejoining is strictly dependent on the integrity of the NHEJ repair system.
Highlights
It is known that exposure to ionizing radiation (IR) causes many types of DNA damage, and, among these, double-strand breaks (DSBs) are considered the most dangerous threat to genomic integrity [1,2]
The formation and rejoining of DNA DSBs were analyzed by determining the number of ionizing radiation induced foci (IRIF) of c-H2AX and 53BP1 proteins in CCD-34Lu cells irradiated with 0.5 Gy of c-rays
Since our results suggest that DSBs are repaired faster in the G2-phase cells during the first time-window after irradiation (0.5– 6h), we analyzed the kinetics of RAD51 and RPA foci formation, representative of homologous recombination (HR) repair system involvement, in CCD-34Lu cells irradiated with 5 Gy (Figure 5)
Summary
It is known that exposure to ionizing radiation (IR) causes many types of DNA damage, and, among these, double-strand breaks (DSBs) are considered the most dangerous threat to genomic integrity [1,2]. It has been demonstrated that high-LET radiation induces clusters of DNA lesions along the particle track while low-LET radiation causes sparse ionizations. When administered at high doses, lowLET radiation can lead to complex DNA damage [3] consisting of DSBs associated with base damages as well as non-DSB damage clusters comprised of base lesions, apyrimidinic or apurinic sites and single-strand breaks that can produce additional DSBs due to damage processing [4]. The efficiency of DNA repair after exposure to IR depends on the complexity of the radio-induced damage [5]. After exposure to IR, the extensive phosphorylation of histone H2AX at
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