Involvement of MEIG1 in the NHEJ DNA Repair Pathway

Introduction

Double strand breaks (DSBs) are deleterious to the cell and can cause cell death, or lead to genomic instability, a hallmark of cancerous processes, if not properly repaired. The Non Homologous End Joining (NHEJ) DNA repair mechanism is the default repair pathway in the cell. During the G2 phase of the cell cycle the homologous recombination (HR) mechanism can operate as well. MEIG1 (meiosis-expressed gene) is a protein known for its importance to spermatogenesis, as Meig1 knockout (KO) male mice cannot produce mature sperm, and thus are infertile. The very few sperm cells that could be obtained from the epididymis of KO mice, had a highly fragmented chromatin, suggesting that MEIG1 plays a role in maintaining genome integrity during spermatogenesis. In this study, we show that the MEIG1 protein has a more general role and is important for efficient repair through the NHEJ pathway.

Materials and Methods

To evaluate MEIG1`s general role in the DSB repair process we first monitored the intracellular localization of MEIG1 upon exposure to genotoxic treatment, in various somatic cell lines. Next, we generated Meig1-KO NIH-3T3 fibroblasts, through the CRISPR/Cas9 system, and compared NHEJ efficiency of these cells to that of WT cells. We followed the repair kinetics by monitoring the characteristic recruitment and disappearance of 53BP1 foci in NHEJ, after exposure to the genotoxic drug phleomycin. A plasmid expressing EGFP was used to monitor NHEJ repair efficiency in cells.

Results and Discussion

In somatic cells, we noticed that MEIG1 is readily recruited to the nucleus upon exposure to genotoxic drugs (etoposide), or in response to UV radiation, as has been shown in known DNA repair proteins. This suggests that MEIG1`s function in DNA repair is not restricted to germ cells. By following the recruitment and disappearance of 53BP1 foci after exposure to the drug phleomycin, we found a significantly delayed appearance and disappearance of foci in the MEIG1-deficient cells, clearly suggesting delayed kinetics of repair. When an EGFP-based system was applied to monitor repair efficiency, we found that DNA repair was significantly less efficient in MEIG1-deficient cells. Expression of recombinant MEIG1 in these Meig1-KO cells restored repair efficiency to WT levels.

Conclusions

Given these results, we concluded that MEIG1 plays a role in the DNA repair process and that its absence results in delayed repair kinetics and reduced efficiency of repair by the NHEJ pathway.