CHK2 regulates MUS81-dependent DSBs in response to replication stress and BRCA2 deficiency.

MUS81 is a structure-specific endonuclease that processes DNA intermediates during mitosis and in S-phase following replication stress. It plays a crucial role in cleaving deprotected reversed forks in BRCA2-deficient cells. However, how MUS81 is regulated during replication stress in human cells remains unknown. Our study reveals that CHK2 binds to the MUS8-EME2 complex in S-phase through the FHA domain and positively regulates the formation of DSBs in response to persistent or pathological replication stress. Mechanistically, our cellular and biochemical data identify a dual-site phosphorylation regulatory mechanism involving priming at the CDK2 site S95 followed by CHK2-dependent modification of the activatory site S97. Phosphorylation of MUS81 does not affect recruitment to the stalled forks but is crucial for the replication stress-dependent association with SLX4 in S-phase. At deprotected forks, in BRCA2-depleted cells, the CHK2-MUS81 complex assembles downstream of fork reversal and degradation, and CHK2-dependent phosphorylation is essential for replication fork recovery and viability. Together, our findings elucidate a novel regulatory mechanism of the MUS81 complex in S-phase and reveal a previously unrecognized role of the ATM-CHK2 axis in responding to persistent replication fork arrest or fork deprotection in the absence of BRCA2.