Fig 2: Rad50 dimerization is essential for ATM activation. A, schematic diagram of wild-type Rad50, mutant Rad50 containing the homodimerization domain FKBP replacing the zinc hook domain, and the coiled-coils deletion mutant. B, elution profiles of Rad50 (FKBP) and Nbs, in the presence or absence of the rapamycin analog AP20187 (100 nm) after incubation of MR (FKBP) and Nbs1, by gel filtration. Fractions from the Superose 6 separations were analyzed by SDS-PAGE and analyzed by Western blotting using antibodies directed against Rad50 and Nbs1. C, kinase assays as in Fig. 3C except with 30 nm wild-type MR, MR(C2G), and MR(FKBP). D, kinase assays as in C but with varying amounts of AP20187 (25, 50, 100, 200, and 400 nm).

Fig 3: FBXW7 interacts with the MRN complex and induces its degradation by lysosomes.A COS-7 cells were transiently transfected with pCDNA3 Flag FBXW7 and nuclear (NE) and cytosolic extracts (S100) immunoprecipitated with anti-Flag or normal mouse serum (IgG) as a control. Immunoprecipitated materials were analyzed by Western blotting. Inp: the input lane was loaded with 1/20 of the extract used in each assay. Data are representative of two independent experiments. B Similar to A, but using anti-MRE11, anti-NBS1, or anti-RAD50 antibodies. Data are representative of two independent experiments. C Nuclear extracts of U2OS cells were used to immunoprecipitate endogenous FBXW7, and the obtained complexes were analyzed by immunoblotting. IgG: immunoprecipitation using normal rabbit serum, as a control. Inp: the input lane was loaded with 1/20 of the nuclear extract. Western blot against PLK1 was used as a control of the efficiency of FBXW7 immunoprecipitation. Data are representative of three independent experiments. D U2OS cells were treated with Con A (for the indicated times), NH4Cl (24 h), rapamycin (Rapa, 16 h) or trehalose (Tre, 24 h), whole cell extracts or nuclear and cytosolic extracts (where indicated) prepared, and membranes immunoblotted with the indicated antibodies. C: extracts of untreated cells. Western blots are representative of at least three replicates. E Whole cell extracts from U2OS or transduced U2OS::HA FBXW7 or U2OS::HA FBXW7?F cells were electroblotted and probed with the indicated antibodies. Western blot against cyclin E was used as a control of the efficiency of FBXW7 or FBXW7?F expression. F U2OS::HA FBXW7 and U2OS::HA FBXW7?F cells were treated or not with Con A (8 h) and analyzed by immunoblotting. *non-specific band from a previous immunoblotting. Data from E and F are representative results from three independent experiments. Quantitative fold change in proteins was determined relative to the loading control (a actinin or ß actin).

Fig 4: Lollipop schematic diagram of RAD50 LOF mutation distribution in (a) familial ESCC cases, (b) sporadic ESCC cases, and (c) controls from Henan. * stands for stop gain mutation.

Fig 5: Schematic model of MRN in the absence of ATP (open) or the presence of ATP (closed), based on structural analysis of bacterial and archaea Mre11/Rad50 complexes (6, 8). Rad50 is shown as light gray globular domains containing Walker A (A) and Walker B (B) motifs connected by intramolecular, antiparallel coiled-coils (CC), that attach to each other through the zinc hook motifs (Zn hook). Stars indicate the approximate positions of the ATP-binding residues in Rad50. Mre11 is shown as dark gray globular domains representing the nuclease/dimerization domain and the capping domain, connected to the linker that binds to the coiled-coils of Rad50. The closed state is associated with a higher affinity for DNA ends and with DNA tethering, while the movement from the closed to the open state is required for Mre11 nuclease activity and promotion of resection.5 The zinc hook is shown attached here, but the complex has also been observed with hooks unattached, both in solution and bound to DNA (30).