Human DNA2/BLM and EXO1 participate in parallel long-range resection pathways for repair of DSB due to replication stress

Double stranded breaks (DSB) are lesions repaired by the homologous recombination (HR) pathway during S/G2 phase. MRN/CtIP nuclease activity initiates a short 5' end resection followed by a longer resection to generate a recombinogenic 3' ssDNA sufficient to activate a cell cycle checkpoint and ensure HR. It is postulated that the long-range resection occurs via two pathways. One requires BLM helicase and the other utilizes EXO1. Here we show that DNA2 nuclease may function with BLM to effect the long-range 5' resection. Using camptothecin to induce DSBs, we have shown that DNA2 and EXO1 function in two parallel pathways. In the absence of both DNA2 and EXO1, DNA resection is markedly impaired in comparison to instances where DNA2 and EXO1 are depleted individually. S-phase checkpoint is probably impaired due to diminished resection, RPA and Chk1 phosphorylation. Cells also exhibit reduced RPA2, RAD51 and ssDNA foci after S-phase specific DNA damage. Moreover, DNA2 and EXO1 knockdown cells have increased DNA-PK phosphorylation, suggesting that interference with HR may activate NHEJ. In clonogenic assays, DNA2 and EXO1 knockdown cells are sensitive to DNA damaging agents camptothecin and cisplatin. Overall, we demonstrate that DNA2/BLM is important for HR in human cells undergoing replication stress