DNA damage-dependent nuclear dynamics of the Mre11 complex

The Mre11 complex has been implicated in diverse aspects of the cellular re sponse to DNA damage. We used in situ fractionation of human fibroblasts to carry out cytologic analysis of Mre11 complex proteins in the double-stran d break (DSB) response. In situ fractionation removes most nucleoplasmic pr otein, permitting immunofluorescent localization of proteins that become mo re avidly bound to nuclear structures after induction of DNA damage. We fou nd that a fraction of the Mre11 complex was bound to promyelocyte leukemia protein bodies in undamaged cells. Within 10 min after gamma irradiation, n uclear retention of the Mre11 complex in small granular foci was observed a nd persisted until 2 h postirradiation. In light of the previous demonstrat ion that the Mre11 complex associated with ionizing radiation (IR)-induced DSBs, we infer that the protein retained under these conditions was associa ted with DNA damage. We also observed increased retention of Rad51 followin g IR treatment, although IR induced Rad51 foci were distinct from Mre11 foc i. The ATM kinase, which phosphorylates Nbs1 during activation of the S-pha se checkpoint, was not required for the Mre11 complex to associate with DNA damage. These data suggest that the functions of the Mre11 complex in the DSB response are implicitly dependent upon its ability to detect DNA damage .