DNA double-strand breaks (DSBs) are the most deleterious damage which cells can encounter. Unrepaired or mis-repaired DSBs can result in genomic instability and cell death. Therefore, DSBs pose a serious threat to genome integrity. Two main repair pathways, canonical non-homologous end-joining (c-NHEJ) and homologous recombination (HR), are known to play a primary role in the repair of DSBs. Cell cycle-specific studies have revealed that c-NHEJ is active throughout the cell cycle, whereas, HR is active in the late-S and G2 phase where a sister chromatid is available as a template for repair. Molecular characterization of these pathways has discovered various key players, such as Ku70/80 and DNA-PKcs of c-NHEJ and Rad51, BRCA2 and Rad54 of HR. Recent findings have made it evident that when c-NHEJ or HR is impaired, alternative end-joining (alt-EJ) pathway operates to remove the DSBs. While alt-EJ acts as a global rescuing mechanism, the removal of DSBs by alt-EJ comes at a cost of elevated chromosomal translocations and sequence alteration at the break ends. Factors implicated in alt-EJ are Mre11, CtIP, DNA ligase 1/3 and PARP1. Recently, DNA polymerase theta (Polθ) was shown to promote alt-EJ by annealing the micro-homologies (MHs), present internal to the resected break ends. Therefore, this pathway is also referred to as DNA polymerase theta-mediated end-joining (TMEJ).

Most of the knowledge about the mechanistic details and the factors involved in HR comes from the studies performed with yeast (S. cerevisiase). In S. cerevisiase, Rad52 was discovered as the key HR player whose absence results in defects in DNA repair, increased sensitivity to IR and cell death. Surprisingly, loss of Rad52 in vertebrate cells has no effect on DNA repair and Rad52 knock-out mice are fertile and viable. However, increasing evidences suggest that Rad52 is involved in HR in mammalian cells. Earlier work performed in the laboratory of Prof. Löbrich showed that Rad52-GFP foci peak in the late G2 phase and persist in the consequent M phase. A study published by Feng et al. (2011) showed that, in BRCA2-deficient mammalian cells, inactivation of Rad52 is synthetically lethal for the cells. However, the exact function of Rad52 in BRCA2-proficient as well as deficient cells is not yet clearly understood.

In this study the function of Rad52 during DSB repair in G2 phase mammalian cells was characterized. In contrast to earlier speculations, in HeLa cells it was shown that Rad52 is not involved in the loading of Rad51 on to the resected 3'-ssDNA overhangs in G2 phase cells and, thus, cannot compensate for the loss of BRCA2. A synthetically lethal relationship was observed between BRCA2 and Rad52, indicating an important role for Rad52 in BRCA2-depleted HeLa and 82-6 (fibroblast) cells. Importantly, by using HeLa-Rad52-GFP cells, it was shown for the first time that BRCA2-depleted cells show significantly increased amounts of Rad52-GFP foci in G2 phase cells. yH2AX foci analysis, however, showed that the increased numbers of Rad52-GFP foci do not imply activation of a Rad52-dependent alternative repair pathway. Interestingly, co-depletion of BRCA2 and Rad52 rescued the BRCA2 repair defect. Furthermore, it was observed that the rescue of the BRCA2 repair defect was due to the activation of the Polθ-mediated TMEJ repair pathway, which gave rise to increased numbers of chromosomal fusions. These results were true for both Hela and fibroblast (82-6 & HSC-62) cells.

Notably, in HeLa cells, it was shown that the two resection-dependent pathways, HR & TMEJ, are active simultaneously in G2 phase. Nevertheless, rescue of the BRCA2-repair defect after depleting Rad52 was still observed in HeLa cells.

In conclusion, the results suggest that due to its ss-DNA binding activity, Rad52 binds to the resected 3'-ssDNA overhangs in BRCA2-deficient cells. This binding of Rad52 prevents TMEJ from repairing the resected DSB ends as repair by TMEJ can result in increased chromosomal fusions. Thus, by preventing the action of TMEJ pathway, Rad52 suppresses the formation of chromosomal fusions and maintains genomic stability in BRCA2-deficient cells. In context to cancer therapy, inactivation of Rad52 in BRCA2-deficient tumors can prove to be a potential therapeutic strategy. Furthermore, in this study it was shown that inactivation of Polθ and BRCA2 results in significantly increased numbers of chromosomal fusions in HeLa cells. This result is consistent with earlier published data where it was shown that inactivation of Polθ in BRCA2-deficient tumors increases chromosomal aberrations and enhances cell killing. Therefore, combined inactivation of Rad52 and Polθ might prove to be more potent and, thus, provide an alternative strategy to specifically kill BRCA2-deficient cancer cells.