The Essential Function of the MRN Complex in the Resolution of Endogenous Replication Intermediates

The MRN complex (Mre11/Rad50/Nbs1) is important in double-strand break (DSB) recognition, end resection, replication fork stabilization, and ATM and ATR activation. Complete deletion of MRN is incompatible with cell and organism life, presumably due to replication-born DSBs; however, the underlying mechanism remains unknown. We devised a noninvasive high-content assay, termed high-content microscopy-assisted cell-cycle phenotyping (hiMAC), to investigate the fate of cells lacking Nbs1. Surprisingly, deletion of Nbs1 does not kill cells during replication. The primary lesions in Nbs1-deleted cells are replication intermediates that result from defective resolution rather than fork destabilization. These lesions are converted to DSBs in the subsequent G2 phase, which subsequently activate Chk1, delay G2 progression, and lead to chromosome instability. Nbs1-deleted cells establish a DSB equilibrium that permits cell cycling but activates p53, causing G1 and G2 arrest, and cell death. Thus, we identify a physiological role of Nbs1 in the resolution of stalled replication forks.