| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 2041470 | Cell Reports | 2014 | 14 Pages |
•Nbs1 null does not kill mammalian cells during replication•Nbs1 mutants initially accumulate replication intermediate, not DSBs•Chk1 and p53 differentially regulate cell-cycle arrest and death of Nbs1-null cells•The hiMAC assay enables DNA damage response profiling during cell-cycle progression
SummaryThe 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.
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