Discussion
We have shown that EXD2 facilitates DSB resection thus promoting recruitment of RPA and homologous recombination. Accordingly, cells depleted for EXD2 show spontaneous chromosomal instability and are sensitive to DNA damage induced by agents that generate DSBs. Furthermore, we establish that EXD2 functionally interacts with the MRN complex utilizing its 3′-5′ exonuclease activity to accelerate DSB resection and promote efficient HR. In line with this, complementation experiments showed that exonuclease-dead mutant protein failed to complement these phenotypes. Interestingly, EXD2 seems to be dispensable for the initial sensing of the break as evidenced by efficient γH2AX and CHK2 phosphorylation, and most likely acts downstream of MRE11. Finally we reveal that both, EXD2 and MRE11 function in the same pathway for DSB resection and HR. It is unclear at present why cells would need two exonucleases with the same polarity. However, a paradigm for such a requirement is evident from the fact that cells have two alternate machineries, consisting of BLM-DNA2-RPA-MRN and EXO1-BLM-RPA-MRN that carry out long-range resection9, 10, 13, 44. Thus, by analogy EXD2 may function together with MRE11 to accelerate resection in the 3′-5′ direction in order to efficiently produce short 3′ ssDNA overhangs. This could promote faster generation of longer stretches of ssDNA, which in turn may serve as a better substrate for BLM–DNA2 or BLM-EXO1 to initiate long-range resection. Accordingly, depletion of EXD2 adversely impacts on this process. Ultimately, efficient generation of ssDNA with minimal homology length required for productive HR would supress unscheduled deleterious recombination events. This may be particularly important in vertebrates, as they require significantly longer stretches of ssDNA (200-500 bp) to initiate productive HR 45, which is in contrast to yeast, where as little as 60 bp of 3′ ssDNA is sufficient to support HR 46. Not mutually exclusive is the possibility that EXD2 could also augment resection efficiency under specific circumstances, for instance in the presence of modifications to the damaged DNA and/or polypeptides bound at the 5′ ends. In line with this, we show that in vivo EXD2 depletion impairs short-range resection. Recently, it has been proposed that MRE11 may create multiple incisions on the DNA strand undergoing resection up to 300 bp distal to the break, which could allow for more efficient resection 21, 22. Indeed, inhibition of MRE11’s endonuclease activity seems to be dominant in promoting the generation of ssDNA over its exonuclease activity 23. Thus, we postulate a model whereby EXD2 functionally collaborates with the resection machinery, most likely utilizing DNA nicks generated by MRE11’s endonuclease activity 3′ of the DSB. This would enhance the generation of ssDNA tails required for efficient homologous recombination (model Fig. 6d).
In summary, our work identifies EXD2 as a critical factor in the maintenance of genome stability through homologous recombination dependent repair of DSBs, including those induced by commonly used anti-cancer agents, such as IR or CPT. This highlights EXD2 itself and/or its enzymatic activity as a potential candidate for development of anti-cancer drugs.