Accurate chromosome segregation during mitosis is temporally and spatially coordinated by fidelity-monitoring checkpoint systems. Deficiencies in these checkpoint systems can lead to chromosome segregation errors and aneuploidy, and promote tumorigenesis. Here, we report that the TRAF-interacting protein (TRAIP), a ubiquitously expressed nucleolar E3 ubiquitin ligase important for cellular proliferation, is localized close to mitotic chromosomes. Its knockdown in HeLa cells by RNA interference (RNAi) decreased the time of early mitosis progression from nuclear envelope breakdown (NEB) to anaphase onset and increased the percentages of chromosome alignment defects in metaphase and lagging chromosomes in anaphase compared with those of control cells. The decrease in progression time was corrected by the expression of wild-type but not a ubiquitin-ligase-deficient form of TRAIP. TRAIP-depleted cells bypassed taxol-induced mitotic arrest and displayed significantly reduced kinetochore levels of MAD2 (also known as MAD2L1) but not of other spindle checkpoint proteins in the presence of nocodazole. These results imply that TRAIP regulates the spindle assembly checkpoint, MAD2 abundance at kinetochores and the accurate cellular distribution of chromosomes. The TRAIP ubiquitin ligase activity is functionally required for the spindle assembly checkpoint control.

DNA double-strand breaks (DSBs) affect cell survival and genomic integrity. They are repaired by a highly coordinated process called the DNA damage response. Here, we report that the ubiquitously expressed nucleolar E3 ubiquitin ligase TRAF-interacting protein (TRAIP), previously shown to regulate the spindle assembly checkpoint, has an essential role during the DNA damage response. A biotinylation proximity screening assay (BioID) identified Ku80, Ku70, SMARCA5 (SNF2H) and DNA-PKcs as novel TRAIP interactors. Co-immunoprecipitations demonstrated that the interaction of TRAIP with Ku80 was transiently increased while the one with SMARCA5 was strongly decreased after treatment of HeLa cells with neocarzinostatin (NCS). Treatment of fibroblasts from a microcephalic primordial dwarfism patient carrying a hypomorphic TRAIP mutation or shRNA-mediated knockdown of TRAIP in HeLa cells with NCS impaired the activation of ataxia-telangiectasia mutated (ATM), a protein kinase crucial for the DNA damage response. As consequence, the maintenance of γH2AX and Chk2-T68 phosphorylation, two downstream targets of ATM, was significantly abrogated after NCS-inflicted DSBs. DNA repair assays showed that TRAIP inhibits incorrect end utilization during non-homologous end joining. These observations highlight TRAIP as novel regulator of ATM activity in DNA damage signaling.