A phosphorylation switch at MRE11 links ATM-ATR and calcium signaling to safeguard stalled replication fork stability.

MRE11 safeguards genome stability at stalled replication forks, where its activity must be tightly controlled to prevent nascent strand DNA degradation (NSD). However, the upstream signaling mechanisms that limit NSD remain poorly defined. Here, we identify Ser649 (S649) as a previously unrecognized phosphorylation site that limits MRE11 association with stalled forks. We show that S649 phosphorylation is robustly induced by replication stress or elevated cytosolic calcium levels, and is mediated by the calcium-responsive CaMKK2-AMPKα axis in concert with ATR, but independently of CHK1. Loss of S649 phosphorylation enhances MRE11 binding to DNA and increases its association with stalled forks, driving excessive NSD, elevated DNA damage, and increased sensitivity to PARP inhibition. We find that the ATM-mediated S676/S678 phosphorylation primes S649 phosphorylation, which in turn facilitates subsequent phosphorylation of SQ/TQ sites in MRE11. Moreover, we find that CaMKK2-AMPKα activation requires ATR but is independent of ATM. Collectively, our findings reveal a hierarchical signaling mechanism that couples calcium signaling with ATM/ATR pathways to prevent NSD at stalled forks and preserve genome integrity.